Beta-Hydroxyisovaleryl-Shikonin Eradicates Epithelial Cell Adhesion Molecule-Positive Liver Cancer Stem Cells by Suppressing dUTP Pyrophosphatase Expression.

Cancer stem cells (CSCs) play an essential role in tumorigenesis, chemoresistance, and metastasis. Previously, we demonstrated that the development of hepatocellular carcinoma (HCC) is dictated by a subset of epithelial cell adhesion molecule-positive (EpCAM+) liver CSCs with the activation of Wnt signaling. In this study, we evaluated the expression of dUTP pyrophosphatase (dUTPase), which plays a central role in the development of chemoresistance to 5-fluorouracil, in EpCAM+ HCC cells. We further evaluated the effect of beta-hydroxyisovaleryl-shikonin (ß-HIVS), an ATP-noncompetitive inhibitor of protein tyrosine kinases, on HCC CSCs. EpCAM and dUTPase were expressed in hepatoblasts in human fetal liver, hepatic progenitors in adult cirrhotic liver, and a subset of HCC cells. Sorted EpCAM+ CSCs from HCC cell lines showed abundant nuclear accumulation of dUTPase compared with EpCAM-negative cells. Furthermore, treatment with the Wnt signaling activator BIO increased EpCAM and dUTPase expression. In contrast, ß-HIVS treatment decreased dUTPase expression. ß-HIVS treatment decreased the population of EpCAM+ liver CSCs in a dose-dependent manner in vitro and suppressed tumor growth in vivo compared with the control vehicle. Taken together, our data suggest that dUTPase could be a good target to eradicate liver CSCs resistant to 5-fluorouracil. ß-HIVS is a small molecule that could decrease dUTPase expression and target EpCAM+ liver CSCs.

Effect of adoptive T-cell immunotherapy on immunological parameters and prognosis in patients with advanced pancreatic cancer.

BACKGROUND AIMS: Immunotherapy is effective for many types of cancer, but its benefits in advanced pancreatic cancer, which has a poor prognosis, are not well established. In this study, the authors examined the effects of adoptive T-cell immunotherapy (ATI) on immune cell profiles and prognosis in patients with unresectable advanced pancreatic cancer. METHODS: Seventy-seven patients with unresectable advanced pancreatic cancer were treated with six cycles of αß T cells alone or in combination with chemotherapy or chemoradiation. Immune cell profiles in peripheral blood samples obtained before and after treatment were comprehensively evaluated by flow cytometry. Furthermore, associations between changes in immune cell frequencies and prognosis were determined. RESULTS: ATI prolonged survival to 18.7 months compared with previous estimates of 6.2-11.1 months for patients treated with chemotherapy alone. ATI decreased CD3+CD4+CD8- T cell frequency in peripheral blood and increased CD3+CD4-CD8+ T cell frequency. An increase in CD3+ T cells and CD3+TCRγδ- T cells in peripheral blood after treatment was associated with a good prognosis. CONCLUSIONS: ATI altered the immune profile in peripheral blood, including CD3+CD4-CD8+ T cells, and improved prognosis in pancreatic cancer.

Effects of adaptive immune cell therapy on the immune cell profile in patients with advanced gastric cancer.

BACKGROUND: Immunotherapy for cancer patients has been the subject of attention in recent years. In this study, we investigated whether αßT-cell therapy causes changes in the host's immune cell profile, and if so, the effect of these changes on prognosis. METHODS: Peripheral blood mononuclear cells (PBMCs) from 30 gastric cancer patients who had completed one course of αßT-cell therapy were analyzed. The peripheral blood immune cell profile was established using PBMCs by counting the frequency of CD4+ helper T cells, CD8+ killer T cells, regulatory T cells (Tregs), and myeloid-derived suppressor cells and measuring the expression of their surface markers. The changes after treatment and their association with response to treatment were investigated. RESULTS: Immune cell profiles changed greatly after treatment. The frequency of CD4+ helper T cells decreased, but that of CD8+ killer T cells increased. The frequency of programmed cell death 1 (PD-1)+ effector Tregs increased significantly, but only in the non-progressive disease (non-PD) group, in which it was significantly higher compared with the PD group. Patients in whom the frequency of PD-1+ effector Tregs increased had a significantly better prognosis than those in whom it decreased. CONCLUSION: Our results suggested that αßT-cell therapy changes the host's immune cell profile, and an increase in PD-1+ effector Tregs may help improve prognosis.

Inactivation of Transcriptional Repressor Capicua Confers Sorafenib Resistance in Human Hepatocellular Carcinoma.

BACKGROUND & AIMS: Sorafenib is a multireceptor tyrosine kinase inhibitor that can prolong overall survival in patients with advanced hepatocellular carcinoma (HCC). Although most HCC patients who receive sorafenib ultimately show disease progression, it still is unclear whether and how HCC cells acquire chemoresistance during sorafenib treatment in human beings. METHODS: We analyzed surgically resected HCC tissues from a patient who received sorafenib for prevention of HCC recurrence after surgery (Adjuvant Sorafenib for Hepatocellular Carcinoma after Resection or Ablation trial) and established patient-derived HCC cells. Whole-exome sequence analysis was performed to detect mutations in sorafenib-resistant clones. We examined 30 advanced HCC cases immunohistochemically and 140 HCC cases enrolled in the Adjuvant Sorafenib for Hepatocellular Carcinoma after Resection or Ablation trial using microarray analysis to evaluate the association of Capicua Transcriptional Repressor (CIC) status with sorafenib treatment response. RESULTS: We found a CIC mutation in recurrent HCC specimens after sorafenib. CIC encodes Capicua, a general sensor of receptor tyrosine kinase signaling. HCC cells established from the recurrent tumor specimen showed chemoresistance to sorafenib in vitro and in vivo. Established sorafenib-resistant Huh1 and Huh7 cell lines showed reduced expression of Capicua without mutations. Immunohistochemical analysis showed that HCC patients with low Capicua expression showed poor overall survival. Microarray analysis showed that the CIC gene signature could predict the preventive effect of adjuvant sorafenib treatment on HCC recurrence. Intriguingly, although CIC knockdown induced sorafenib resistance in HCC cell lines, regorafenib suppressed growth of sorafenib-resistant, Capicua-inactivated HCC cells and inhibited extracellular signal-regulated kinase phosphorylation. CONCLUSIONS: Evaluation of Capicua status may be pivotal to predict response to sorafenib, and regorafenib treatment could be effective to treat HCC with functional Capicua impairment.

A Novel mTOR Inhibitor; Anthracimycin for the Treatment of Human Hepatocellular Carcinoma.

BACKGROUND/AIM: Anthracimycin, a secondary metabolite of Streptomyces, has been shown to inhibit the invasion of certain cancer cell lines. MATERIALS AND METHODS: In this study we evaluated the effect of anthracimycin on cell growth and signaling pathways in hepatocellular carcinoma (HCC). RESULTS: Anthracimycin suppressed cell proliferation and motility and induced apoptosis in human HCC cell lines. Furthermore, anthracimycin had no effect on the enrichment of EpCAM-high liver cancer stem cells (CSCs), while fluorouracil dramatically enriched the CSCs with activation of the stemness-related genes EPCAM and SOX9 in HuH7 cells. Mechanistically, anthracimycin suppressed mammalian target of rapamycin (mTOR) signaling, and was most effective at inhibiting HCC cell proliferation with mTOR activation. CONCLUSION: Anthracimycin is a novel mTOR inhibitor capable of suppressing the proliferation of CSCs and non-CSCs equally well in HCC, and it is suggested that anthracimycin could be effective in the eradication of HCC associated with mTOR-signaling activation.

De Novo Emergence of Mesenchymal Stem-Like CD105+ Cancer Cells by Cytotoxic Agents in Human Hepatocellular Carcinoma.

BACKGROUND: Cancer stem cells (CSCs) are considered a pivotal target for the eradication of hepatocellular carcinoma (HCC). Recently, we reported that the CSC markers epithelial cell adhesion molecule (EpCAM) and CD90 are expressed independently in primary HCCs and cell lines, and CD90+ cells share features of metastatic vascular endothelial cells and express the vascular endothelial marker CD105, a co-receptor of transforming growth factor-beta. METHODS: The EpCAM+ cell lines HuH1 and HuH7 were treated with 5-fluorouracil (5-FU) or epirubicin in vitro. Gene and protein expression levels were evaluated by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and fluorescence-activated cell sorting, respectively. The expression of CD105 in primary HCC was evaluated by immunohistochemistry. The relationship of CD105 expression status and HCC prognosis was evaluated using 85 surgically resected HCC tissues by Kaplan-Meier survival analysis. RESULTS: 5-FU or epirubicin treatment resulted in the generation of CD90+ and CD105+ cells in vitro in HuH1 and HuH7 cells, which originally contain no CD90+ or CD105+ cells. This phenomenon was validated by qRT-PCR analysis with activation of the epithelial-mesenchymal transition (EMT) program regulators Snail family zinc finger 1 (SNAI1) and SNAI2. Immunohistochemical analysis indicated that CD105+ cells were morphologically identical to vascular endothelial cells in untreated primary HCCs. However, surgically resected specimens after transcatheter arterial chemoembolization clearly indicated that CD105+ cancer cells survived at the peripheral edge of the tumor. Kaplan-Meier survival analysis indicated that HCCs expressing CD105 showed poor prognosis after surgery with statistical significance. CONCLUSIONS: Taken together, our data highlight the role of CD105+ HCC cells with activation of the EMT program generated de novo after cytotoxic therapy on the prognosis of HCC patients.

TSU-68 ameliorates hepatocellular carcinoma growth by inhibiting microenvironmental platelet-derived growth factor signaling.

BACKGROUND: TSU-68 is a multikinase inhibitor that targets platelet-derived growth factor receptors (PDGFRs). In the present study, we evaluated the effect of TSU-68 on the tumor-microenvironment interaction in hepatocellular carcinoma (HCC). MATERIALS AND METHODS: HCC and fibroblast cell lines (HuH7, Hep3B, HuH1 and WI-38) were used to evaluate their interactions. Cancer characteristics were evaluated by spheroid formation and tumorigenicity in immunodeficient mice. Time-lapse image analysis was performed to monitor cell motility. RESULTS: Although PDGFA was abundantly expressed, PDGFR-α was predominantly located in the cytoplasm and was not functional in HuH7 cells. Co-culture experiments demonstrated that HCC cells induced phosphorylation of PDGFR-α in WI-38 fibroblasts and that stimulated fibroblasts, in turn, boosted the spheroid formation capacity of HCC cells. TSU-68 inhibited phosphorylation of PDGFR-α in WI-38 cells and suppressed the growth of subcutaneously co-injected HuH7/WI-38 tumor xenografts. CONCLUSION: TSU-68 inhibits stromal PDGF signaling activated by cancer cells and suppresses HCC growth.