Intrahepatic Metastasis Of Hepatocellular Carcinoma Research Articles

Cancer-Associated Fibroblasts Promote Vascular Invasion of Hepatocellular Carcinoma via Downregulating Decorin-integrin β1 Signaling.

Hepatocellular carcinoma (HCC) is a common malignancy worldwide, and the high ratio of recurrence and metastasis remains the main cause of its poor prognosis. Vascular invasion of HCC includes microvascular invasion (MVI) and portal vein tumor thrombosis (PVTT) and is regarded as a common roadmap of intrahepatic metastasis in HCC. However, the molecular mechanism underlying vascular invasion of HCC is largely unknown. Here, we analyzed the transcriptomes of primary tumors, PVTT tissues, and tumor tissues with or without MVI. We found that extracellular matrix-related pathways were involved in vascular invasion of HCC and that decorin secreted by cancer-associated fibroblasts was gradually downregulated from normal to tumor tissues and more so in PVTT tissues. We also established that low-level decorin expression is an independent risk factor for MVI and it is associated with a poor prognosis. Decorin downregulated integrin β1 and consequently inhibited HCC cell invasion and migration in vitro. Co-staining DCN and integrin β1 revealed that DCN dynamically regulated integrin β1 protein expression. Integrin β1 knockdown significantly inhibited HCC invasion and migration, and decorin combined with such knockdown synergistically augmented the anti-metastatic effects. Co-IP assay confirmed the direct interaction of decorin with integrin β1. Our findings showed that targeting cancer-associated fibroblast-related decorin is not only a promising strategy for inhibiting HCC vascular invasion and metastasis but also provides insight into the clinical treatment of patients with PVTT.

MiR-631 Inhibits Intrahepatic Metastasis of Hepatocellular Carcinoma by Targeting PTPRE.

MicroRNAs (miRNAs) have been reported to play critical roles in the pathological development of hepatocellular carcinoma (HCC), one of the most common cancers in the world. Our study aims to explore the expression, function and mechanism of miR-631 in HCC. Our findings are that expression of miR-631 is significantly down-regulated in HCC tissue compared with that in adjacent non-cancerous tissue, and low expression of miR-631 in HCC tissue is associated with cirrhosis, multiple tumors, incomplete tumor encapsulation, poor tumor differentiation, and high TNM stage. Our test results showed that miR-631 could inhibit migration, invasion, epithelial–mesenchymal transition (EMT) and intrahepatic metastasis of HCC. Receptor-type protein tyrosine phosphatase epsilon (PTPRE) as a downstream target of miR-631 could promote migration, invasion and EMT of HCC cells. Besides, the expression of PTPRE had a negative correlation with the expression of miR-631 both in vivo and in vitro, and increasing expression of PTPRE could reverse inhibitory effects of miR-631 in HCC cells. In sum, our study first demonstrated that miR-631 targeted PTPRE to inhibit intrahepatic metastasis in HCC. We gain insights from these findings into the mechanism of miRNAs regulation in HCC metastasis and further introduce a novel therapeutic target for HCC treatment.

Intrahepatic metastases may be specific to hepatocellular carcinoma due to the coagulation and fibrinolytic systems (Review).

Hepatocellular carcinoma (HCC) is different from other solid tumors because it is commonly associated with the occurrence of intrahepatic metastasis. Additionally, the liver, unlike other organs, is the main site of coagulation and fibrinolytic factor production. Therefore, it was speculated that coagulation and fibrinolytic factors could be associated with intrahepatic metastasis of HCC. Do the coagulation and fibrinolytic systems protect HCC cells against anoikis during infiltration and metastasis? Conversely, do the coagulation and fibrinolytic systems lead to immune escape of HCC cells by affecting the immune microenvironment of patients? The current review aimed to present a number of novel hypotheses for the treatment of HCC by exploring the mechanisms of coagulation and fibrinolytic factors in the regulation of cancer growth.

Animal model of intrahepatic metastasis of hepatocellular carcinoma: establishment and characteristic

One of the most important and striking characteristics of hepatocellular carcinoma (HCC) with intrahepatic metastasis, is that it results in extremely poor prognosis. Animal models have become a fundamental and very useful in research for disease study. However, some limitation has arisen from these model systems. We have therefore established a model of HCC with intrahepatic metastasis and noticed some differential appearances in different HCC cell lines. Luciferase-transfected HCC cell lines MHCC97-H and PLC/PRF/5 were inoculated into SCID mice via spleen. Observation the intrahepatic metastasis by bioluminescence imaging in vivo and comparing of the differential formation of metastatic lesions between different HCC cell lines by incorporating physical anatomy was done. Animal models for HCC intrahepatic metastasis were well established. However, there were some clearly noticed differences between MHCC97-H and PLC/PRF/5 cell lines. The group of MHCC97-H cell line readily metastasis in the liver, whereas group PLC/PRF/5 cell line developed extensive intrahepatic metastasis and formed large tumor in situ in the spleen. MHCC97-H and PLC/PRF/5 cell lines can be used to successfully establish a model of HCC intrahepatic metastasis with distinctive characteristics, which provides an important direction for the study of the mechanism of HCC intrahepatic metastasis, and may hopefully provide a basis for clinical treatment.

A Novel lncRNA NONHSAT053785 Acts as an Independent Risk Factor for Intrahepatic Metastasis of Hepatocellular Carcinoma.

PurposeLong noncoding RNAs (lncRNAs) in body fluids have been considered as promising novel biomarkers for tumor-related diseases. The present study aimed to investigate the expression level of lncRNA NONHSAT053785 in serum and its correlation with clinical characteristics of hepatocellular carcinoma (HCC) patients.MethodsThe droplet digital PCR (ddPCR) was used to measure the serum levels of NONHSAT053785 in 112 HCC patients, 96 chronic hepatitis B (CHB) patients, and 99 healthy controls (HC). The correlation between NONHSAT053785 and clinical characteristics was analyzed by chi-square test and Spearman correlation test. The risk factors of intrahepatic metastasis (IM) were detected by univariate and multivariate analyses. Furthermore, the diagnostic value of NONHSAT053785 in HCC and its predictive ability in IM were evaluated by the receiver operating characteristic (ROC) curves.ResultsThe level of NONHSAT053785 was significantly increased in the serum of HCC patients and was higher in HCC patients with IM as compared to those without. Additionally, the expression level of NONHSAT053785 was significantly related to IM, Child–Pugh classification, and peripheral blood indicators such as liver metabolic enzymes and positively correlated to IM, Barcelona Clinic Liver Cancer (BCLC) staging, and some peripheral blood indicators. Furthermore, the serum NONHSAT053785 was indicated as an independent predictor for IM in the elderly, non-smoking, drinking, and tumor size ≥5 cm subjects. The area under the ROC curve (AUC) was 0.801 (P <0.0001) for diagnosis of HCC and 0.678 (P =0.0015) for predicting IM.ConclusionThe increase in serum NONHSAT053785 levels was related to an increased risk of IM, and hence, may serve as a novel biomarker for the diagnosis of HCC and the prediction of IM.

The tumor diameter cut-off for predicting microscopic intrahepatic metastasis of hepatocellular carcinoma patients without treatment history differs from that of hepatocellular carcinoma patients with a treatment history.

Intrahepatic metastasis (IM) of hepatocellular carcinoma (HCC) occurs via vascular invasion; the tumor diameter that affects the risk of micro intra-hepatic metastasis (MIM) should be larger than that which affects the risk of micro vessel invasion (MVI). The aim of the present study was to determine the optimum tumor diameter cut-off value for predicting the presence of MIM in HCC patients without treatment history and HCC patients with a treatment history and to compare these diameters between cases of MVI and MIM. This retrospective study included 621 patients without macroscopic vessel invasion or intrahepatic metastasis on preoperative imaging who underwent hepatectomy. The cut-off tumor diameter for predicting the presence of MIM was determined by a receiver operating characteristic curves analysis. The optimum cut-off value for predicting the presence of MIM in HCC patients without treatment history was 43mm. In contrast, the optimum cut-off value for predicting the presence of MIM in HCC patients with a treatment history was 20mm. Among 46 HCC patients with MIM without treatment history, there were 20 patients with MIM without MVI who were considered to have potential multi-centric (MC) tumors rather than IM. The cumulative overall survival rates in patients with MIM without MVI (potential MC) was significantly better than that in patients with both MIM and MVI (P = 0.022). The tumor diameter cut-off value for predicting MIM differed between HCC patients without treatment history and with a treatment history and slightly smaller than those for predicting MVI beyond our expectation.

Research on the relationship between the expression of mitofusin 2 and phosphatidylinositol transfer membrane-associated protein 3 in intrahepatic metastasis of hepatocellular carcinoma

Objective To investigate the relationship between expression of mitofusin 2 (Mfn2) and phosphatidylinositol transfer membrane-associated protein 3 (PITPNM3) in the intrahepatic metastasis in hepatocellular carcinoma (HCC). Methods A total of 149 patients with HCC patients who underwent hepatectomy were enrolled in this study. The clinical follow-up data and pathological tissue sections were collected. Immunohistochemical staining was performed for Mfn2 and PITPNM3 in pathological section. The relationship between the expression of Mfn2 and PITPNM3 immunohistochemical staining scores and clinicopathological data in patients were analyzed. Results In the negative expression group of Mfn2, 54.55% patients’ PITPNM3 expressions are strongly positive. In the strongly positive expression group of Mfn2, 52.17% patients’ PITPNM3 expressions are negative. The expression of Mfn2 is highly negative correlated with PITPNM3 expression; Patients have a high histological grade, high rate of intrahepatic metastasis, late clinical stage and bad prognosis with high expression of PITPNM3, 56.34% patients are intrahepatic metastasis; Patients have a relatively low histological grade, low rate of intrahepatic metastasis, early clinical stage and good prognosis with high expression of Mfn2, only 14.08% patients are intrahepatic metastasis. Conclusion Mfn2 may be a key protein that inhibits intrahepatic metastasis of HCC. Key words: Mitofusin 2; Phosphatidylinositol transfer membrane-associated protein 3; Hepatocellular carcinoma; Intrahepatic metastasis

Amplification of SMYD3 promotes tumorigenicity and intrahepatic metastasis of hepatocellular carcinoma via upregulation of CDK2 and MMP2.

SMYD3, a member that belongs to the SET and MYND-domain (SMYD) family, has also been proven to largely participate in gene transcription regulation and progression of several human cancers as a histone lysine methyltransferase. However, the role and significance of SMYD3 in both the clinic and progression of hepatocellular carcinoma (HCC) remain unclear. Herein, we find that SMYD3 is increased in cirrhotic livers, and strikingly upregulated in hepatocellular carcinoma (HCC) tissues and cell lines. Subsequent analyses suggest that high expression level of SMYD3 significantly correlates with the malignant characteristics of HCC, and predicts poor prognosis in patients. Our results show that overexpression of SMYD3 increases, while silencing of SMYD3 inhibits, cell proliferation, invasiveness and tumorigenicity both in vitro and in vivo. SMYD3 also promotes intrahepatic metastasis of HCC cells. For the mechanisms, we identify that SMYD3 bound to CDK2 and MMP2 promoter and increased H3K4me3 modification at the corresponding promoters to promote gene transcription. Importantly, pharmacological targeting of SMYD3 with BCI-121 inhibitor effectively repressed the tumorigenicity of HCC cells. Finally, our results show that gene locus amplification is a cause for SMYD3 overexpression in HCC. These findings not only uncover that SMYD3 overexpression promotes the tumorigenicity and intrahepatic metastasis of HCC cell via upregulation of CDK2 and MMP2, but also suggest SMYD3 could be a practical prognosis marker or therapeutic target against the disease.

Expression and clinical value of anti-differentiation antagonizing noncoding RNA in patients with intrahepatic metastasis of hepatocellular carcinoma

Objective To explore the expression and clinical significance of the long non-coding RNA anti-differentiation antagonizing noncoding RNA (DANCR) in the patients with intrahepatic metastasis of hepatocellular carcinoma (HCC). Methods The tumor tissues, pre- and post-operative plasma samples of 24 HCC intrahepatic metastasis patients undergoing resection, the tumor tissues and plasma samples of 24 HCC patients, and the plasma samples of 24 healthy volunteers were collected from the Central Hospital of Wuhan between Jan. 2015 and Oct. 2017. The expression of DANCR mRNA level was detected by quantitative real-time quantitative polymerase chain reaction (PCR) in the tumor tissues and plasma samples. The relationship of DANCR mRNA expression with clinicopathological characteristics was analyzed. Results The expression levels of DANCR mRNA in the tumor tissues (2.85±0.23 vs. 1.63±0.16, P<0.01) and the plasma (0.043±0.007 vs. 0.018±0.005, P<0.01) of the patients with intrahepatic metastasis of HCC were significantly higher than those of HCC patients. In addition, the DANCR mRNA expression was significantly lower after surgical resection than that in patients with intrahepatic metastasis of HCC before surgery (0.035±0.006 vs. 0.012±0.004, P<0.05). The expression of DANCR mRNA in tumor tissues of patients with HCC intrahepatic metastasis was correlated with the size of primary tumor (P<0.05), chronic hepatitis B history (P<0.05) and Child-Pugh grade (P<0.05). Conclusion DANCR expression is closely related to the development of HCC. Key words: Carcinoma, hepatocellular; Anti-differentiation; Noncoding RNA; Intrahepatic metastasis

Establishment of Highly Intrahepatic Metastatic Cell Line of HCC by In vivo Selection and Investigation of Mechanism by Integrated Microarray Analysis

Introduction: Intrahepatic metastasis is a frequent pattern of tumor recurrence in hepatocellular carcinoma (HCC). The mechanism of intrahepatic metastasis was speculated as following; cancer cells invaded portal vein and spread via portal vein flow. However, the molecular mechanism of intrahepatic metastasis remains unclear. Here, we investigated the molecular mechanism of intrahepatic metastasis in HCC by establishing a novel highly metastatic cell line and analyzing the integrated microarray analysis. Methods: Highly intrahepatic metastatic HCC cell line (HuH7-M) was established by in vivo selection using trans-portal vein metastatic model. Briefly, parentalHuH-7-Luc cells (HuH7-P) were injected into the spleen of SCID Beige male mouse. After six weeks from the injection, liver metastatic tumors were dissociated and expanded in culture medium. The cell populations were subjected to the next cycle. This cycle was repeated in four cycles and the novel highly metastatic cell line (HuH7-M) was established. The characteristics of HuH7-P and HuH7-M were compared by MTS assay, transendothelial migration assay, sphere formation assay, and Annexin V analysis. Tumorigenesis was compared by simultaneously administering each cell to spleen followed by splenectomy. Subsequently, integrated microarray analysis was performed by combination with mRNA expression and miRNA expression analysis. Results: After four cycles, the metastatic tumor size and the tumorigenesis rate were gradually increased in HuH7-M (33% vs 88%, p=0.036). The microscopic finding revealed that the differentiation of tumor was not changed. Proliferative potential was increased in HuH7-M. But, invasive potential, migration potential, cancer stemness, and morphological change were not observed. The cell of HuH7-M showed the lower rate of apoptosis (3.4% vs 13.4%). Tumorigenesis assay showed that intrahepatic metastatic tumor was formed only in HuH7-M (67% vs 0%). Integrated microarray analysis demonstrated a specific combination of mRNA and miRNA related to apoptosis as a potential target. Conclusion: Highly intrahepatic metastatic HCC model was established by in vivo selection, and the result of integrated microarray analysis indicated a molecular mechanism of intrahepatic metastasis in HCC mediated by apoptosis.834_A Figure 1. The tumorigenesis rate were gradually increased by in vivo selection834_B Figure 2. HuH7-M showed the lower rate of apoptosis than HuH7-P

Incidental intraoperative multiple bile duct adenomas found in conjunction with hepatocellular carcinoma

Literature reviews from 1999-2016 determined bile duct adenoma (BDA) to be a very rare benign tumor. BDA usually presents as a single nodular lesion, most often found incidentally and is very difficult to distinguish from hepatocellular carcinoma (HCC) on imaging. We present a case of multiple BDAs in a patient with a known HCC. Patient is a 65-year-old male, non-cirrhotic, cured hepatitis C after Harvoni treatment, with one 4.0 cm lesion found on CT in segment 6. Due to the difficulty of diagnosis based on CT and MRI, biopsy was performed and confirmed HCC. Intra-operatively four other nodular lesions, in addition to the 4.0 cm HCC, were found involving both lobes of the liver surface. Sizes varied from 3 to 7 mm, intraoperative ultrasound did not view other lesions. Intraoperative diagnosis was one HCC with four other small HCCs, possible HCC intrahepatic metastasis. Partial liver resection with several liver wedge resections was performed, all lesions were resected and the patient recovered well. Pathology results showed 4cm HCC in segment 6 and four small (< 1cm) BDAs of segments 2, 3, 6, and 7. BDA is a rare benign liver tumor, usually a small (< 5mm) singular lesion and rarely presents together with HCC. Our case showed multiple BDAs can coexist with HCC. From our literature search, this has never been reported. This is the first case of multiple BDAs in conjunction with HCC. Increased knowledge and awareness is essential to appropriately diagnose and treat patients.

PO-184 Role of ubiquitin-conjugating enzymes in chromosome instability and breast cancer metastasis

IntroductionLab-on-a-chip (LOC) systems can present cells multiple cues that cells are subjected to, including gradients of cytokines and secreted proteins from neighbouring cells, biochemical and mechanical interactions with the extracellular matrix, and direct cell-to-cell contacts in a controllable and reproducible fashion. Simulating microenvironment with LOC devices is crucial to understand metastasis preference of cancer cells especially for planning personalised therapies in hepatocellular carcinoma (HCC).Material and methodsPeristaltic-pump was used to circulate HCC cells, HuH-7 and SNU-449. Survival under shear stress was analysed by fluorometric and microscopic analysis. Adhesion and proliferation were measured by live cell analysis system. HUVEC cells were used to simulate extravasation of circulating cells. BEAS2B, HS-27A and THLE-2 cells were used as homing targets to simulate bone, lung and liver, respectively. Image analysis was performed with fluorescent and confocal microscopy.Results and discussionsFirstly, we determined the effect of shear stress in a time-dependent manner on adhesion and proliferation of HCC cells. Circulation under shear stress up to 30’ caused an increase in both adhesion and proliferation significantly, whereas longer exposure time has a negative effect on adhesion, proliferation and, survival. HuH-7 cells with epithelial phenotype found to gain increased survival capacity under shear stress than its mesenchymal counterpart SNU-449. Then, we investigated the homing preference of HCC cells by using a novel micro-circulation LOC device. Shear stress, microenvironmental conditions (medium, matrigel, coating, etc.), cell labels and numbers (homing, HCC) were optimised for LOC. Representation of endothelial barrier was succeeded by monolayer coating of ’flow-channel’ by HUVEC cells. HCC cells were circulated for 4 hours then LOCs were incubated for 3 days. The extravasation and homing preference of HCC cells were determined by confocal imaging. Consistent with the high frequency of intrahepatic metastasis in HCC, SNU-449 cells mostly preferred to home into the channel with normal liver cells.ConclusionOur study suggests designed LOC system has a potential to predict homing capacity and organ preference of circulating tumour cells in HCC. In addition to precision medicine applications such as drug screening, this device could be used as a three-dimensional tool for understanding tumor-microenvironment interactions with mechanistic explanations in metastasis.

The NCX1/TRPC6 Complex Mediates TGFβ-Driven Migration and Invasion of Human Hepatocellular Carcinoma Cells

TGFβ plays an important role in the progression and metastasis of hepatocellular carcinoma (HCC), yet the cellular and molecular mechanisms underlying this role are not completely understood. In this study, we investigated the roles of Na+/Ca2+ exchanger 1 (NCX1) and canonical transient receptor potential channel 6 (TRPC6) in regulating TGFβ in human HCC. In HepG2 and Huh7 cells, TGFβ-stimulated intracellular Ca2+ increases through NCX1 and TRPC6 and induced the formation of a TRPC6/NCX1 molecular complex. This complex-mediated Ca2+ signaling regulated the effect of TGFβ on the migration, invasion, and intrahepatic metastasis of human HCC cells in nude mice. TGFβ upregulated TRPC6 and NCX1 expression, and there was a positive feedback between TRPC6/NCX1 signaling and Smad signaling. Expression of both TRPC6 and NCX1 were markedly increased in native human HCC tissues, and their expression levels positively correlated with advancement of HCC in patients. These data reveal the role of the TRPC6/NCX1 molecular complex in HCC and in regulating TGFβ signaling, and they implicate TRPC6 and NCX1 as potential targets for therapy in HCC.Significance: TGFβ induces the formation and activation of a TRPC6/NCX1 molecular complex, which mediates the effects of TGFβ on the migration, invasion, and intrahepatic metastasis of HCC. Cancer Res; 78(10); 2564-76. ©2018 AACR.

Incidence of Occult Intrahepatic Metastasis in Hepatocellular Carcinoma Treated With Transplantation Corresponds to Early Recurrence Rates After Partial Hepatectomy.

This study aimed to compare the incidence of radiologically unrecognized (occult) hepatocellular carcinoma (HCC) lesions in explant hepatectomy specimens from orthotopic liver transplants (OLTs) performed for HCC with rates of HCC intrahepatic recurrence after resection. Resection of HCC is associated with high rates of intrahepatic HCC recurrence. However, it is unclear whether these recurrences represent incomplete resection of unrecognized metastatic lesions from the primary tumor or subsequent de novo tumor formation due to inherent biological proclivity for HCC formation. We collected patient, tumor, and pathology data on HCC patients treated surgically from 3696 OLTs in the Organ Procurement and Transplantation (OPTN) national database, 299 OLTs at a single transplant center, and 232 partial hepatectomies from a hepatobiliary cancer center. In the OPTN and high-volume transplant center cohorts, 37% and 42% of patients had occult HCC lesions on explant pathology, respectively. Among cancer center patients, the 2-year recurrence rate was 46%, and 74% of patients who recurred presented with liver only recurrence. Although the transplant and resection populations differ, occult multifocality is common in transplant explants and similar to the 46% early recurrence rate following partial hepatectomy. These data suggest that noncurative resection often results from occult intrahepatic multifocality present at the time of resection rather than a malignant predisposition of the remnant liver with de novo tumorigenesis.

Overexpression of a disintegrin and metalloproteinase 21 is associated with motility, metastasis, and poor prognosis in hepatocellular carcinoma

Cell motility plays an important role in intrahepatic metastasis of hepatocellular carcinoma (HCC), and predicts poor prognosis in patients. The present study investigated the role of a disintegrin and metalloproteinases (ADAMs) in HCC, since these proteins are known to be associated with cell motility. We confirmed the expression of 12 ADAMs with putative metalloproteinase activity in HCC cells, and established a KYN-2 HCC cell line stably expressing short interfering RNA against ADAM21 to investigate the effect of ADAM21 deficiency on HCC cell motility and metastasis in vitro and in vivo. We also examined ADAM21 expression in a cohort of 119 HCC patients by immunohistochemistry. ADAM21 was overexpressed in KYN-2 cells, and its knockdown reduced invasion, migration, proliferation, and metastasis relative to controls. In clinical specimens, ADAM21 positivity was associated with vascular invasion, large tumor size, high histological grade, and lower overall and recurrence-free survival as compared to cases that were negative for ADAM21 expression. A multivariate analysis revealed that ADAM21 positivity was an independent risk factor for overall (P = 0.003) and recurrence-free (P = 0.001) survival. These results suggest that ADAM21 plays a role in HCC metastasis and can serve as a prognostic marker for disease progression.

Peri-tumor associated fibroblasts promote intrahepatic metastasis of hepatocellular carcinoma by recruiting cancer stem cells

Fibroblasts have been reported to play an important role in hepatocellular carcinoma (HCC). However, the role of fibroblasts have not been fully understood. Conditioned medium collected from human peri-tumor tissue-derived fibroblasts (CM-pTAFs) showed high metastasis ability than human HCC tissues-derived fibroblasts (CM-TAFs). To determine what component was secreted from fibroblasts, we used Bio-Plex analysis system and compared the factors secreted from CM-pTAFs and CM-TAFs, found a series of up-regulated cytokines in the CM-pTAFs, including IL-6, CCL2, CXCL1, CXCL8, SCGF-β, HGF and VEGF. Pretreatment of IL-6 inhibitor Tocilizumab could inhibit metastasis the HCC cell treated with CM-pTAFs in vitro and in vivo. The expression of CCR2 and CXCR1 were up-regulated after CM-pTAFs treatment in HCC cell line SMMC-7721. Flow cytometric analysis experiment showed that most CCR2 or CXCR1 positive cells were also EpCAM positive. In vitro studies also showed that CM-pTAFs could increase stemness of SMMC-7721. In addition, neutralization of SCGF-β and HGF could significantly reduce metastasis and viability of cancer stem cells treated with CM-pTAFs. Taken together, these results indicated that the peri-tumor tissues derived fibroblasts may promote development of HCC by recruiting cancer stem cells and maintaining their stemness characteristic.

主病巣の完全自然壊死後に肝内転移巣のみ残存した肝細胞癌の一例

今回われわれは主病巣の完全自然壊死後に肝内転移巣のみ残存した非常に稀な肝細胞癌症例を経験した．症例は59歳，男性．アルコール性肝障害および十二指腸潰瘍で近隣病院にてフォローを受けていたが，CT検査で肝S8に1.5 cmの結節影が出現し当院に紹介となった．肝EOB-MRI検査およびダイナミックCT検査で動脈相で濃染，門脈相でwash outされる1.5 cmの結節像をS8に認めた．肝細胞癌と診断し肝S8亜区域切除術を施行した．病理組織学的所見では，結節は周囲との境界は比較的明瞭で肝細胞癌を疑う腫瘍性病変と考えられたが，肝細胞が脱落して炎症性肉芽組織に置換されておりviableな腫瘍細胞はみられなかった．その近傍に約0.2 cmの中分化型肝細胞癌の増殖が認められた．1.5 cmの結節は肝細胞癌が自然壊死に至った主病巣であり0.2 cmの肝内転移巣のみが残存したものと考えられた．

Abstract 163: Genetic difference between multicentric carcinogenesis and intrahepatic metastasis of hepatocellular carcinoma

Abstract Multiple hepatocellular carcinoma (HCC) is categorized into 2 types; multicentric hepatocarcinogenesis (MC) and intrahepatic metastasis (IM). Although discrimination of the types of multiple HCC is of clinical importance, it is quite difficult to determine it correctly even after histological diagnosis. In order to classify multiple HCC into MC and IM, we compared pairs of multiple HCC samples from 12 patients who were clinically diagnosed MC, 6 patients with IM with regard to molecular aberrations, and 2 patients with pairs of primary HCC and extrahepatic metastasis such as adrenal grand and lung metastasis. Exome sequence showed that 125 mutations (90.6%) per patient with IM were common, which were consistent with the result of a pair of primary HCC and extrahepatic metastasis. On the other hand, 138 somatic mutations per tumor, and no common somatic mutations were identified in the 7 patients with MC. Especially only a few passenger mutations were different between IM specimens suggesting that IM pairs developed from the common ancestor. In contrast, 5 patients who clinically diagnosed MC had common mutations (41.7%), which indicates that pairs of HCC of these patients were genetically IM. Notably, mutation signatures obtained from exome sequence data of 2 nodules from the same patients with IM were similar, while those from patients with MC were different by profiling analysis. On the other hand, methylation profile shows that 2 HCC samples from the same patients with MC as well as IM were classified into the same cluster; the methylation status from the different ancestor is similar, and not altered through carcinogenesis. Taken together, comparison of mutations in a pair of HCCs makes it possible to classify multiple HCCs into MC and IM, which is difficult to be correctly determined in clinical practice, and is available to make treatment plans for multiple HCC patients. Furthermore, methylation status of 2 nodules of MC from same patient were similar, while those of mutation signatures were different, which suggests that the epigenomic changes are the earlier event prior to the genomic alterations. Citation Format: Yutaka Midorikawa, Shogo Yamamoto, Kenji Tatsuno, Hiroki Ueda, Shingo Tsuji, Genta Nagae, Tadatoshi Takayama, Hiroyuki Aburatani. Genetic difference between multicentric carcinogenesis and intrahepatic metastasis of hepatocellular carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 163.

Abstract 689: Downregulation of TIMP2 via HIF-1á/miR-210/HIF-3á regulatory feedback circuit enhances cancer metastasis in hepatocellular carcinoma

Abstract Cancer metastasis is a multi-step process that involves a series of tumor-stromal interaction, including extracellular matrix (ECM) remodeling which requires a concerted action of multiple proteolytic enzymes and their endogenous inhibitors. This study investigated the role of tissue inhibitor of metalloproteinases 2 (TIMP2) in the context of hepatocellular carcinoma (HCC) metastasis. We found that TIMP2 was the most significantly downregulated member among the TIMP family in human HCCs. Moreover, TIMP2 underexpression was frequent (41.1%; 23/56) in human HCCs as compared to the corresponding non-tumorous livers and was significantly associated with direct liver invasion into the adjacent liver parenchyma and poorer survival outcomes of the HCC patients. Furthermore, stable silencing of TIMP2 in HCC cell lines enhanced the cell invasive ability and ECM degradation associated with formation of invadopodia-like feature, suggesting that TIMP2 is a negative regulator of HCC metastasis. Furthermore, using orthotopic tumor xenograft model, we demonstrated that ectopic expression of TIMP2 open reading frame in the highly metastatic HCC cell line MHCC-97L not only significantly reduced the incidence of tumor microsatellite formation and venous invasion in the primary hepatic tumor xenografts, but also pulmonary metastasis, suggesting that both extrahepatic and intrahepatic metastasis in HCC was suppressed by TIMP2 expression. Mechanistically, TIMP2 suppression in a hypoxic environment was induced through a regulatory feedback circuit consisting of HIF-1á, miR-210 and HIF-3á. Taken altogether, our findings established that TIMP2 was frequently downregulated in human HCCs and its downregulation was associated with aggressive behavior and poorer patients’ outcome. Its suppression was under the regulation of a novel feedback circuit consisting of HIF-1á/ miR-210/ HIF-3á. Overall, our study has provided solid in vitro and in vivo evidence that TIMP2 is an important regulator of ECM degradation and HCC metastasis. These findings demonstrated that perturbation of the dynamic HIF-1a signaling circuit by miR-210 inhibitor abolished TIMP2 downregulation and suppressed HCC cell invasion. They also support the notion that targeting against HIF-1á signaling is a promising direction to tackle the hypoxic responses in HCC elicited by transarterial chemo-embolization (TACE) treatment to HCC patients. Citation Format: Alan KL Kai, Lo Kong Chan, Regina CL Lo, Joyce Lee, Carmen CL Wong, Jack CM Wong, Irene OL Ng. Downregulation of TIMP2 via HIF-1á/miR-210/HIF-3á regulatory feedback circuit enhances cancer metastasis in hepatocellular carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 689.

Pokemon promotes the invasiveness of hepatocellular carcinoma by enhancing MEF2D transcription.

Pokemon, a master oncogene crucial for the tumorigenicity and progression of a variety of cancers, has been demonstrated to enhance the proliferation and survival of hepatocellular carcinoma (HCC). However, the contribution of Pokemon to the invasiveness of HCC has not yet been studied. In this study, we employed HCC cells to investigate the role of Pokemon in the invasion of HCC with multidisciplinary approaches. Pokemon overexpression was found to be closely associated with invasion and intrahepatic metastasis of HCC in clinical specimens. Suppression of Pokemon attenuated the invasion of HCC cells by in vitro transwell and wound-healing assays. Myocyte enhancer factor 2D (MEF2D), an oncogene that can promote the invasiveness of HCC, was found to be underexpressed during Pokemon silencing in HCC cells. Restoration of MEF2D abolished the effect of Pokemon downregulation on the migration of HCC cells. Further experiments verified that Pokemon binds two putative recognition sites located within the upstream region of the MEF2D promoter and enhances its transcription. The association between Pokemon and MEF2D was further confirmed in HCC specimens. Animal experiments further confirmed that Pokemon downregulation attenuated the metastasis of HCC cells in mice. Collectively, Pokemon was found to enhance the migration and invasion of HCC by increasing MEF2D expression. Thus, targeting Pokemon and MEF2D may be an effective strategy to suppress the metastasis of HCC.