The evolving concept of liver cancer stem cells

Liver cancer is an often fatal malignant tumor with a high recurrence rate and chemoresistance. The major malignant phenotypes of cancer, including recurrence, metastasis, and chemoresistance, are related to the presence of cancer stem cells (CSCs). In the past few decades, CSCs have been identified and characterized in many tumors including liver cancer. Accumulated evidence has revealed many aspects of the biological behavior of liver CSCs and the mechanism of their regulation. Based on these findings, a number of studies have investigated eradication of liver CSCs. This review focuses on the recent advances in our understanding of the biology of liver CSCs and the development of strategies for their treatment

Defeating EpCAM+ liver cancer stem cells by targeting chromatin remodeling enzyme CHD4 in human hepatocellular carcinoma

13301甲第4446号博士（医学）金沢大学博士論文要旨Abstract 以下に掲載：Journal of Hepatology 63(5) pp.1164-1172 2015. ELSEVIER. 共著者：Kouki Nio, Taro Yamashita, Hikari Okada, Mitsumasa Kondo, Takehiro Hayashi, Yasumasa Hara, Yoshimoto Nomura, Sha Sha Zeng, Mariko Yoshida, Tomoyuki Hayashi, Hajime Sunagozaka, Naoki Oishi, Masao Honda, Shuichi Kanek

The evolving concept of liver cancer stem cells

Liver cancer is an often fatal malignant tumor with a high recurrence rate and chemoresistance. The major malignant phenotypes of cancer, including recurrence, metastasis, and chemoresistance, are related to the presence of cancer stem cells (CSCs). In the past few decades, CSCs have been identified and characterized in many tumors including liver cancer. Accumulated evidence has revealed many aspects of the biological behavior of liver CSCs and the mechanism of their regulation. Based on these findings, a number of studies have investigated eradication of liver CSCs. This review focuses on the recent advances in our understanding of the biology of liver CSCs and the development of strategies for their treatment. © 2017 The Author(s)

Oncostatin M renders epithelial cell adhesion molecule-positive liver cancer stem cells sensitive to 5-fluorouracil by inducing hepatocytic differentiation

金沢大学附属病院消化器内科Recent evidence suggests that a certain type of hepatocellular carcinoma (HCC) is hierarchically organized by a subset of cells with stem cell features (cancer stem cells; CSC). Although normal stem cells and CSCs are considered to share similar self-renewal programs, it remains unclear whether differentiation programs are also maintained in CSCs and effectively used for tumor eradication. In this study, we investigated the effect of oncostatin M (OSM), an interleukin 6-related cytokine known to induce the differentiation of hepatoblasts into hepatocytes, on liver CSCs. OSM receptor expression was detected in the majority of epithelial cell adhesion molecule-positive (EpCAM+) HCC with stem/progenitor cell features. OSM treatment resulted in the induction of hepatocytic differentiation of EpCAM+ HCC cells by inducing signal transducer and activator of transcription 3 activation, as determined by a decrease in stemness-related gene expression, a decrease in EpCAM, α-fetoprotein and cytokeratin 19 protein expressions, and an increase in albumin protein expression. OSM-treated EpCAM+ HCC cells showed enhanced cell proliferation with expansion of the EpCAM-negative non-CSC population. Noticeably, combination of OSM treatment with the chemotherapeutic agent 5-fluorouracil (5-FU), which eradicates EpCAM-negative non-CSCs, dramatically increased the number of apoptotic cells in vitro and suppressed tumor growth in vivo compared with either saline control, OSM, or 5-FU treatment alone. Taken together, our data suggest that OSM could be effectively used for the differentiation and active cell division of dormant EpCAM+ liver CSCs, and the combination of OSM and conventional chemotherapy with 5-FU efficiently eliminates HCC by targeting both CSCs and non-CSCs. ©2010 AACR

Jagged1 DNA Copy Number Variation Is Associated with Poor Outcome in Liver Cancer

Notch signaling abnormalities are reported to be involved in the acceleration of malignancy in solid tumors and stem cell formation or regeneration in various organs. We analyzed specific genes for DNA copy number variations in liver cancer cells and investigated whether these factors relate to clinical outcome. Chromosome 20p, which includes the ligand for Notch pathways, Jagged1, was found to be amplified in several types of hepatoma cells, and its mRNA was up-regulated according to α-fetoprotein gene expression levels. Notch inhibition using Jagged1 shRNA and γ-secretase inhibitors produced significant suppression of cell growth in α-fetoprotein–producing cells with suppression of downstream genes. Using in vivo hepatoma models, the administration of γ-secretase inhibitors resulted in reduced tumor sizes and effective Notch inhibition with widespread apoptosis and necrosis of viable tumor cells. The γ-secretase inhibitors suppressed cell growth of the epithelial cell adhesion molecule–positive fraction in hepatoma cells, indicating that Notch inhibitors could suppress the stem cell features of liver cancer cells. Even in clinical liver cancer samples, the expression of α-fetoprotein and Jagged1 showed significant correlation, and amplification of the copy number of Jagged1 was associated with Jagged1 mRNA expression and poor survival after liver cancer surgical resection. In conclusion, amplification of Jagged1 contributed to mRNA expression that activates the Jagged1-Notch signaling pathway in liver cancer and led to poor outcome. © 2016 American Society for Investigative PathologyEmbargo Period 12 month

The transcription factor SALL4 regulates stemness of EpCAM-positive hepatocellular carcinoma

Background & Aims: Recent evidence suggests that hepatocellular carcinoma can be classified into certain molecular subtypes with distinct prognoses based on the stem/maturational status of the tumor. We investigated the transcription program deregulated in hepatocellular carcinomas with stem cell features. Methods: Gene and protein expression profiles were obtained from 238 (analyzed by microarray), 144 (analyzed by immunohistochemistry), and 61 (analyzed by qRT-PCR) hepatocellular carcinoma cases. Activation/suppression of an identified transcription factor was used to evaluate its role in cell lines. The relationship of the transcription factor and prognosis was statistically examined. Results: The transcription factor SALL4, known to regulate stemness in embryonic and hematopoietic stem cells, was found to be activated in a hepatocellular carcinoma subtype with stem cell features. SALL4-positive hepatocellular carcinoma patients were associated with high values of serum alpha fetoprotein, high frequency of hepatitis B virus infection, and poor prognosis after surgery compared with SALL4-negative patients. Activation of SALL4 enhanced spheroid formation and invasion capacities, key characteristics of cancer stem cells, and up-regulated the hepatic stem cell markers KRT19, EPCAM, and CD44 in cell lines. Knockdown of SALL4 resulted in the down-regulation of these stem cell markers, together with attenuation of the invasion capacity. The SALL4 expression status was associated with histone deacetylase activity in cell lines, and the histone deacetylase inhibitor successfully suppressed proliferation of SALL4-positive hepatocellular carcinoma cells. Conclusions: SALL4 is a valuable biomarker and therapeutic target for the diagnosis and treatment of hepatocellular carcinoma with stem cell features. © 2013 European Association for the Study of the Liver

Serum cytokine profiles predict survival benefits in patients with advanced hepatocellular carcinoma treated with sorafenib: A retrospective cohort study

金沢大学先進予防医学研究科Background: Sorafenib is a multiple receptor tyrosine kinase inhibitor known to prolong overall survival in patients with advanced hepatocellular carcinoma (HCC). Predicting this drug\u27s survival benefits is challenging because clinical responses are rarely measurable during treatment. In this study, we hypothesized that serum cytokines levels could predict the survival of advanced HCC patients, as sorafenib targets signaling pathways activated in the tumor stromal microenvironment and potentially affects serum cytokine profiles. Methods: Of 143 patients with advanced-stage HCC, 104 who were recruited between 2003 and 2007 received hepatic arterial infusion chemotherapy (HAIC) that mainly targets tumor epithelial cells at S-phase (cohort 1); additionally, 39 recruited between 2010 and 2012 received sorafenib, which primarily targets the stromal vascular endothelial cells. Serum samples were collected and aliquoted prior to the treatment. Serum EGF, bFGF, HGF, IFN-γ, IL-10, IL-12, IL-2, IL-4, IL-5, IL-6, IL-8, IP-10, MIG, PDGF-BB, SCF, SDF1, TGF-β, TGF-α, TNF-α, and VEGF-A were measured via enzyme-linked immunosorbent assays. The Modified Response Evaluation Criteria in Solid Tumors were used to assess tumor responses. Results: The median survival time of HCC patients in cohorts 1 (HAIC-treated) and 2 (sorafenib-treated) were 12.0 and 12.4 months, respectively. Kaplan-Meier analysis revealed no significant survival differences between the 2 groups. Patients who survived more than 2 years after sorafenib treatment exhibited higher serum levels of IL-10, IL-12, TNF-a, IL-8, SDF-1, EGF, PDGF-BB, SCF, and TGF-α. Furthermore, cohort 2 patients with higher serum IL-5 (>12 pg/mL), IL-8 (>10 pg/mL), PDGF-BB (>300 pg/mL), and VEGF-A (>50 pg/mL) levels achieved longer survival; cohort 1 patients did not. Hierarchical cluster analysis of 6 cytokines robustly enriched for comparison analysis between cohorts 1 and 2 (IL-5, IL-8, TGF-α, PDGF-BB, CXCL9, and VEGF-A) revealed that elevation of these cytokines correlated with better survival when treated with sorafenib but not with HAIC. Conclusions: Patients who exhibited survival benefits owing to sorafenib treatment tended to present higher serum cytokines levels, potentially reflecting the activation of stromal signaling in the tumor microenvironment. Our study thus introduces novel biomarkers that may identify advanced HCC patients who may experience survival benefits with sorafenib treatment. © 2017 The Author(s)

肝細胞癌悪性性質獲得に寄与するBMP9分子機構の解明と新規治療法の開発

金沢大学附属病院本研究で我々は、肝細胞癌患者血清中のBMP9値が患者予後に関連する血清バイオマーカーである事を明らかにした。肝がん細胞株を用いた実験により、BMP9はその標的遺伝子であるID1とともに、Wnt/βカテニンシグナルの活性化を介して、EpCAM陽性肝がん幹細胞を活性化する事が明らかとなった。さらに、肝がん細胞株ならびに肝がん移植マウスモデルを用いBMP受容体阻害剤の有用性を検討した結果、BMP9-ID1シグナルを阻害する事で、BMP9による誘導されるEpCAM陽性肝がん幹細胞の活性化が抑制され、細胞増殖や浸潤・遊走といった肝がん悪性形質が有意に抑制される事が明らかとなった。In this study, we aimed to elucidate the role of BMP9 signaling in cancer stem cell (CSC) properties of hepatocellular carcinoma (HCC) and to assess the therapeutic effect of BMP receptor inhibitors in HCC. We have first identified that high BMP9 expression in serum from patients with HCC leads to poorer outcome. BMP9 promoted CSC properties in EpCAM-positive HCC subtype via enhancing ID1 expression in vitro. Additionally, ID1 knockdown significantly repressed BMP9-promoted HCC-CSC properties by suppressing Wnt/β-catenin signaling. Cells treated with BMP receptor inhibitors K02288 and LDN-212854 blocked HCC-CSC activation by inhibiting BMP9-ID1 signaling. Treatment with LDN-212854 suppressed HCC tumor growth by repressing ID1 and EpCAM in vivo. Our study demonstrates the pivotal role of BMP9-ID1 signaling in promoting HCC-CSC properties and the therapeutic potential of BMP receptor inhibitors in treating EpCAM-positive HCC.研究課題/領域番号:18K15807, 研究期間(年度):2018-04-01 – 2021-03-31出典：「肝細胞癌悪性性質獲得に寄与するBMP9分子機構の解明と新規治療法の開発」研究成果報告書　課題番号18K15807（KAKEN：科学研究費助成事業データベース（国立情報学研究所）） （https://kaken.nii.ac.jp/ja/report/KAKENHI-PROJECT-18K15807/18K15807seika/）を加工して作