Effect of respiratory syncytial virus on the growth of hepatocellular carcinoma cell-lines

In several reports, the respiratory syncytial virus (RSV) was identified as an oncolytic virus in cancer cells (e.g., lung and prostate cancer). However, the effects of RSV in hepatocellular carcinoma (HCC) cells have not yet been investigated. Here, we observed the inhibitory effects of RSV infection in HCC cell-lines. Cell growth was significantly decreased by RSV infection in BNL-HCC, Hep3B, Huh-7 and SNU-739 cells. After RSV infection, plaque formation and syncytial formation were observed in affected Hep3B and Huh-7 cells. RSV protein-expression was also detected in Hep3B and Huh-7 cells; however, only Huh-7 cells showed apoptosis after RSV infection. Furthermore, inhibition of cell migration by RSV infection was observed in BNL-HCC, Hep3B, Huh-7 and SNU-739 cells. Therefore, further investigation is required to clarify the molecular mechanism of RSV-mediated inhibition of HCC cell growth, and to develop potential RSV oncolytic viro-therapeutics. [BMB Reports 2015; 48(10): 565-570]     

Transient mTOR inhibition facilitates continuous growth of liver tumors by modulating the maintenance of CD133+ cell populations

The mammalian target of the rapamycin (mTOR) pathway, which drives cell proliferation, is frequently hyperactivated in a variety of malignancies. Therefore, the inhibition of the mTOR pathway has been considered as an appropriate approach for cancer therapy. In this study, we examined the roles of mTOR in the maintenance and differentiation of cancer stem-like cells (CSCs), the conversion of conventional cancer cells to CSCs and continuous tumor growth in vivo. In H-Ras-transformed mouse liver tumor cells, we found that pharmacological inhibition of mTOR with rapamycin greatly increased not only the CD133+ populations both in vitro and in vivo but also the expression of stem cell-like genes. Enhancing mTOR activity by over-expressing Rheb significantly decreased CD133 expression, whereas knockdown of the mTOR yielded an opposite effect. In addition, mTOR inhibition severely blocked the differentiation of CD133+ to CD133- liver tumor cells. Strikingly, single-cell culture experiments revealed that CD133- liver tumor cells were capable of converting to CD133+ cells and the inhibition of mTOR signaling substantially promoted this conversion. In serial implantation of tumor xenografts in nude BALB/c mice, the residual tumor cells that were exposed to rapamycin in vivo displayed higher CD133 expression and had increased secondary tumorigenicity compared with the control group. Moreover, rapamycin treatment also enhanced the level of stem cell-associated genes and CD133 expression in certain human liver tumor cell lines, such as Huh7, PLC/PRC/7 and Hep3B. The mTOR pathway is significantly involved in the generation and the differentiation of tumorigenic liver CSCs. These results may be valuable for the design of more rational strategies to control clinical malignant HCC using mTOR inhibitors.     

Epitope distance to the target cell membrane and antigen size determine the potency of T cell-mediated lysis by BiTE antibodies specific for a large melanoma surface antigen

Melanoma chondroitin sulfate proteoglycan (MCSP; also called CSPG4, NG2, HMW-MAA, MSK16, MCSPG, MEL-CSPG, or gp240) is a surface antigen frequently expressed on human melanoma cells, which is involved in cell adhesion, invasion and spreading, angiogenesis, complement inhibition, and signaling. MCSP has therefore been frequently selected as target antigen for development of antibody- and vaccine-based therapeutic approaches. We have here used a large panel of monoclonal antibodies against human MCSP for generation of single-chain MCSP/CD3-bispecific antibodies of the BiTE (for bispecific T cell engager) class. Despite similar binding affinity to MCSP, respective BiTE antibodies greatly differed in their potency of redirected lysis of CHO cells stably transfected with full-length human MCSP, or with various MCSP deletion mutants and fusion proteins. BiTE antibodies binding to the membrane proximal domain D3 of MCSP were more potent than those binding to more distal domains. This epitope distance effect was corroborated with EpCAM/CD3-bispecific BiTE antibody MT110 by testing various fusion proteins between MCSP and EpCAM as surface antigens. CHO cells expressing small surface target antigens were generally better lysed than those expressing larger target antigens, indicating that antigen size was also an important determinant for the potency of BiTE antibody. The present study for the first time relates the positioning of binding domains and size of surface antigens to the potency of target cell lysis by BiTE-redirected cytotoxic T cells. In case of the MCSP antigen, this provides the basis for selection of a maximally potent BiTE antibody candidate for development of a novel melanoma therapy.     

Microporous cellulosic scaffold as a spheroid culture system modulates chemotherapeutic responses and stemness in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) treatments are evaluated by two-dimensional (2D) in vitro culture systems, despite their limited ability to predict drug efficacy. The three-dimensional (3D) microporous scaffold provides the possibility of generating more reliable preclinical models to increase the efficacy of cancer treatments. The physical properties of a microporous cellulosic scaffold were evaluated. The cellulosic scaffold was biocompatible and had a highly porous network with appropriate pore size, swelling rate, and stiffness of cancer cell cultures. Cellulosic scaffolds were compared with 2D polystyrene for the culture of HepG2 and Huh7 human HCC cells. Cellulosic scaffolds promoted tumor spheroid formation. Cells cultured on scaffolds were more resistant to chemotherapy drugs and showed upregulation of EpCAM and Oct4. The migration ability of HCC cells cultured on scaffolds was significantly greater than that of cells grown in 2D cultures as evidenced by the downregulation of E-cadherin. In addition, the proportion of CD44+/CD133+ HCC cancer stem cells (CSCs) was significantly greater in cells cultured on scaffolds than in those grown in 2D cultures. These findings suggest that cellulosic scaffolds effectively mimic the in vivo tumor behavior and may serve as a platform for the study of anticancer therapeutics and liver CSCs.     

LINC01133 aggravates the progression of hepatocellular carcinoma by activating the PI3K/AKT pathway

LncRNAs exhibit crucial roles in various pathological diseases, including hepatocellular carcinoma (HCC). Therefore, it is significant to recognize the dysregulated lncRNAs in HCC progression. Recently, LINC01133 has been identified in several tumors. However, the biological role of LINC01133 in HCC remains poorly understood. Currently, we focused on the function of LINC01133 in HCC development. We observed that LINC01133 was significantly increased in HCC cells including HepG2, Hep3B, MHCC-97L, SK-Hep-1, and MHCC-97H cells compared with the normal human liver cell line HL-7702. In addition, PI3K/AKT signaling was highly activated in HCC cells. Knockdown of LINC01133 was able to inhibit HCC cell proliferation, cell colony formation, cell apoptosis, and blocked cell cycle arrest in the G1 phase. For another, downregulation of LINC01133 repressed HCC cell migration and invasion. Subsequently, the PI3K/AKT signaling pathway was strongly suppressed by silence of LINC01133 in Hep3B and HepG2 cells. Then, in vivo tumor xenografts models were established using Hep3B cells to explore the function of LINC01133 in HCC progression. Consistently, our study indicated that knockdown of LINC01133 dramatically repressed HCC tumor progression through targeting the PI3K/AKT pathway in vivo. Taken these together, we revealed that LINC01133 contributed to HCC progression by activating the PI3K/AKT pathway.     

The Repressive Effect of miR-148a on TGF beta-SMADs Signal Pathway Is Involved in the Glabridin-Induced Inhibition of the Cancer Stem Cells-Like Properties in Hepatocellular Carcinoma Cells

Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related mortality worldwide. Current standard practices for treatment of HCC are less than satisfactory because of cancer stem cells (CSCs)-mediated post-surgical recurrence. For this reason, targeting the CSCs or the cancer cells with CSCs-like properties has become a new approach for the treatment of HCC. GLA exhibits anti-tumor effects in that it attenuates the proliferation, migration, invasion, and angiogenesis of human cancer cells. However, the functions of GLA in the regulation of CSCs-like properties in HCC cells, and the molecular mechanisms underlying in remain obscure. Here we found that GLA attenuated the CSCs-like properties by the microRNA-148a (miR-148a)-mediated inhibition of transforming growth factor beta (TGF-β)/SMAD2 signal pathway in HCC cell lines (HepG2, Huh-7, and MHCC97H). Indeed, GLA inhibited the activations/expressions of both TGFβ-induced and the endogenous SMAD2. Further, GLA improved the expression of miR-148a in a dose/time-dependent manner. MiR-148a, which targeted the SMAD2-3′UTR, decreased the expression and function of SMAD2. Knockdown of miR-148a abolished the GLA-induced inhibition of TGF-β/SMAD2 signal pathway and the CSCs-like properties in HCC cells. Our study found a novel mechanism that GLA inhibits the CSCs-like properties of HCC cells by miR-148a-mediated inhibition of TGF-β/SMAD2 signal pathway, which may help to identify potential targets for the therapies of HCC.     

Integrin alpha 7 correlates with poor clinical outcomes,and it regulates cell proliferation,apoptosis and stemness via PTK2-PI3K-Akt signaling pathway in hepatocellular carcinoma

This study aimed to evaluate the correlation of integrin alpha 7 (ITGA7) with clinical outcomes and its effect on cell activities as well as stemness in hepatocellular carcinoma (HCC). HCC tumor tissues and paired adjacent tissues from 90 HCC patients were obtained and ITGA7 expression was detected using immunohistochemistry assay. Cellular experiments were conducted to examine the effect of ITGA7 on cell activities, astemness via ITGA7 ShRNA transfection, and compensation experiments were further performed to test whether ITGA7 functioned via regulating PTK2-PI3K-AKT signaling pathway. ITGA7 was overexpressed in tumor tissues compared with paired adjacent tissues and its high expression was correlated with larger tumor size, vein invasion and advanced Barcelona Clinic Liver Cancer stage, and it also independently predicted worse overall survival in HCC patients. In cellular experiments, ITGA7 was upregulated in SMMC-7721, Hep G2, HuH-7 and BEL-7404 cell lines compared with normal human liver cells HL-7702. ITGA7 knockdown suppressed cell proliferation but promoted apoptosis, and it also downregulated CSCs markers (CD44, CD133 and OCT-4) as well as PTK2, PI3K and AKT expressions in SMMC-7721 and Hep G2 cell lines. ITGA7 overexpression promoted cell proliferation but inhibited apoptosis, and it also upregulated CSCs markers in HL-7702 cells. Further compensation experiments verified that ITGA7 regulated cell proliferation, apoptosis and CSCs markers via PTK2-PI3K-Akt signaling pathway. ITGA7 negatively associates with clinical outcomes in HCC patients, and it regulates cell proliferation, apoptosis and CSCs markers via PTK2-PI3K-Akt signaling pathway.     

Prostaglandin E2 receptor EP1 transactivates EGFR/MET receptor tyrosine kinases and enhances invasiveness in human hepatocellular carcinoma cells

Recent evidence indicates that cyclooxygenase-2 (COX-2) and epidermal growth factor receptor (EGFR) are involved in hepatocarcinogenesis. This study was designed to evaluate the possible interaction between the COX-2 and EGFR signaling pathways in human hepatocellular carcinoma (HCC) cells. Immunohistochemical analysis using serial sections of human HCC tissues revealed positive correlation between COX-2 and EGFR in HCC cells (P < 0.01). Overexpression of COX-2 in cultured HCC cells (Hep3B) or treatment with PGE(2) or the selective EP(1) receptor agonist, ONO-DI-004, increased EGFR phosphorylation and tumor cell invasion. The PGE(2)-induced EGFR phosphorylation and cell invasiveness were blocked by the EP(1) receptor siRNA or antagonist ONO-8711 and by two EGFR tyrosine kinase inhibitors, AG1478 and PD153035. The EP(1)-induced EGFR transactivation and cell invasion involves c-Src, in light of the presence of native binding complex of EP(1)/Src/EGFR and the inhibition of PGE(2)-induced EGFR phosphorylation and cell invasion by the Src siRNA and the Src inhibitor, PP2. Further, overexpression of COX-2 or treatment with PGE(2) also induced phosphorylation of c-Met, another receptor tyrosine kinase critical for HCC cell invasion. Moreover, activation of EGFR by EGF increased COX-2 promoter activity and protein expression in Hep3B and Huh-7 cells, whereas blocking PGE(2) synthesis or EP(1) attenuated EGFR phosphorylation induced by EGF, suggesting that the COX-2/PGE(2)/EP(1) pathway also modulate the activation of EGFR by its cognate ligand. These findings disclose a cross-talk between the COX-2/PGE(2)/EP(1) and EGFR/c-Met signaling pathways that coordinately regulate human HCC cell invasion.     

Influence of galectin-9/Tim-3 interaction on herpes simplex virus-1 latency

After HSV-1 infection, CD8(+) T cells accumulate in the trigeminal ganglion (TG) and participate in the maintenance of latency. However, the mechanisms underlying intermittent virus reactivation are poorly understood. In this study, we demonstrate the role of an inhibitory interaction between T cell Ig and mucin domain-containing molecule 3 (Tim-3)-expressing CD8(+) T cells and galectin 9 (Gal-9) that could influence HSV-1 latency and reactivation. Accordingly, we show that most K(b)-gB tetramer-specific CD8(+) T cells in the TG of HSV-1-infected mice express Tim-3, a molecule that delivers negative signals to CD8(+) T cells upon engagement of its ligand Gal-9. Gal-9 was also upregulated in the TG when replicating virus was present as well during latency. This could set the stage for Gal-9/Tim-3 interaction, and this inhibitory interaction was responsible for reduced CD8(+) T cell effector function in wild-type mice. Additionally, TG cell cultures exposed to recombinant Gal-9 in the latent phase caused apoptosis of most CD8(+) T cells. Furthermore, Gal-9 knockout TG cultures showed delayed and reduced viral reactivation as compared with wild-type cultures, demonstrating the greater efficiency of CD8(+) T cells to inhibit virus reactivation in the absence of Gal-9. Moreover, the addition of recombinant Gal-9 to ex vivo TG cultures induced enhanced viral reactivation compared with untreated controls. Our results demonstrate that the host homeostatic mechanism mediated by Gal-9/Tim-3 interaction on CD8(+) T cells can influence the outcome of HSV-1 latent infection, and manipulating Gal-9 signals might represent therapeutic means to inhibit HSV-1 reactivation from latency.     

Concentrations of EpCAM ectodomain as found in sera of cancer patients do not significantly impact redirected lysis and T-cell activation by EpCAM/CD3-bispecific BiTE antibody MT110

Ectodomains of target antigens for antibody-based therapies can be shed from the target cell surface and found in sera of patients. Shed ectodomains of therapeutic targets not only pose the risk of sequestering therapeutic antibodies but, in a multimeric form, of triggering T cell activation by bispecific antibodies binding to CD3 on T cells. Recently, epithelial cell adhesion molecule (EpCAM) has been shown to be activated by release of its ectodomain, called EpEX. Here, we show that only very low amounts of EpEX are detectable in sera of cancer patients. Among 100 cancer patient samples tested, only 17 (17%) showed serum levels of EpEX in excess of 0.05 ng/ml with highest EpEX concentrations of 5.29, 1.37 and 0.52 ng/ml. A recombinant form of human EpEX (recEpEX) was produced to assess its possible effect on redirected lysis and T cell activation by EpCAM/CD3-bispecific BiTE antibody MT110, currently being tested in patients with solid tumor malignancies. RecEpEX had a very minor effect on redirected lysis by MT110 with an approximate IC₅₀ value of 3,000 ng/ml, which is a concentration close to three orders of magnitude higher than the highest EpEX concentration found in cancer patients. Concentrations of 30 ng/ml EpEX in combination with 250 ng/ml MT110 were minimally required to induce a detectable activation of CD4⁺ and CD8⁺ T cells. We conclude that soluble EpEX in sera of cancer patients is unlikely to pose an issue for the efficacy or safety of MT110, and perhaps other antibodies binding to N-terminal epitopes of EpCAM.Key words: EpCAM, T cell, BiTE antibody, immunotherapy, adenocarcinoma, tumor antigen shedding     

Reexpression of ARHI inhibits tumor growth and angiogenesis and impairs the mTOR/VEGF pathway in hepatocellular carcinoma

The Ras-related tumor suppressor gene aplasia Ras homolog member I (ARHI) is frequently downregulated in many types of cancer, including hepatocellular carcinoma (HCC). In this study, we sought to explore the therapeutic implications of ARHI reconstitution in the treatment of HCC. We generated stable cell lines overexpressing ARHI in Hep3B and SK-Hep1 cells, both of which lack endogenous ARHI. The effects of ARHI reexpression on tumor growth and angiogenesis were assessed. Given the key role of mammalian target of rapamycin (mTOR) signaling in HCC progression, we also tested whether ARHI overexpression affected the mTOR pathway. Forced expression of ARHI resulted in a significant inhibition of the proliferation of both Hep3B and SK-Hep1 cells compared to control cells (P<0.01). Cell cycle analysis revealed a G0-G1 arrest induced by ARHI reexpression. Moreover, ARHI reexpression significantly retarded Hep3B xenograft growth in vivo, and caused a marked reduction in tumor angiogenesis assessed by CD31-stained microvessel count. Western blot analysis of the xenografts showed that ARHI overexpression substantially reduced the phosphorylation of two mTOR substrates, S6K1 and 4E-BP1, indicative of an inactivation of the mTOR pathway. Accompanying with the mTOR inactivation, the angiogenic factors, hypoxia-inducible factor 1 alpha and vascular endothelial growth factor, were significantly downregulated. These data highlighted an important role for ARHI in controlling HCC growth and angiogenesis, therefore offering a possible therapeutic strategy against this malignancy.     

Antitumor Efficacy of the Dual PI3K/mTOR Inhibitor PF-04691502 in a Human Xenograft Tumor Model Derived from Colorectal Cancer Stem Cells Harboring a PIK3CA Mutation

PIK3CA (phosphoinositide-3-kinase, catalytic, alpha polypeptide) mutations can help predict the antitumor activity of phosphatidylinositol-3-kinase (PI3K)/mammalian target of rapamycin (mTOR) pathway inhibitors in both preclinical and clinical settings. In light of the recent discovery of tumor-initiating cancer stem cells (CSCs) in various tumor types, we developed an in vitro CSC model from xenograft tumors established in mice from a colorectal cancer patient tumor in which the CD133+/EpCAM+ population represented tumor-initiating cells. CD133+/EpCAM+ CSCs were enriched under stem cell culture conditions and formed 3-dimensional tumor spheroids. Tumor spheroid cells exhibited CSC properties, including the capability for differentiation and self-renewal, higher tumorigenic potential and chemo-resistance. Genetic analysis using an OncoCarta™ panel revealed a PIK3CA (H1047R) mutation in these cells. Using a dual PI3K/mTOR inhibitor, PF-04691502, we then showed that blockage of the PI3K/mTOR pathway inhibited the in vitro proliferation of CSCs and in vivo xenograft tumor growth with manageable toxicity. Tumor growth inhibition in mice was accompanied by a significant reduction of phosphorylated Akt (pAKT) (S473), a well-established surrogate biomarker of PI3K/mTOR signaling pathway inhibition. Collectively, our data suggest that PF-04691502 exhibits potent anticancer activity in colorectal cancer by targeting both PIK3CA (H1047R) mutant CSCs and their derivatives. These results may assist in the clinical development of PF-04691502 for the treatment of a subpopulation of colorectal cancer patients with poor outcomes.     

Galectin-9 induces apoptosis through the calcium-calpain-caspase-1 pathway

Galectin-9 (Gal-9) induced the apoptosis of not only T cell lines but also of other types of cell lines in a dose- and time-dependent manner. The apoptosis was suppressed by lactose, but not by sucrose, indicating that beta-galactoside binding is essential for Gal-9-induced apoptosis. Moreover, Gal-9 required at least 60 min of Gal-9 binding and possibly de novo protein synthesis to mediate the apoptosis. We also assessed the apoptosis of peripheral blood T cells by Gal-9. Apoptosis was induced in both activated CD4(+) and CD8(+) T cells, but the former were more susceptible than the latter. A pan-caspase inhibitor (Z-VAD-FMK) inhibited Gal-9-induced apoptosis. Furthermore, a caspase-1 inhibitor (Z-YVAD-FMK), but not others such as Z-IETD-FMK (caspase-8 inhibitor), Z-LEHD-FMK (caspase-9 inhibitor), and Z-AEVD-FMK (caspase-10 inhibitor), inhibited Gal-9-induced apoptosis. We also found that a calpain inhibitor (Z-LLY-FMK) suppresses Gal-9-induced apoptosis, that Gal-9 induces calcium (Ca(2+)) influx, and that either the intracellular Ca(2+) chelator BAPTA-AM or an inositol trisphosphate inhibitor 2-aminoethoxydiphenyl borate inhibits Gal-9-induced apoptosis. These results suggest that Gal-9 induces apoptosis via the Ca(2+)-calpain-caspase-1 pathway, and that Gal-9 plays a role in immunomodulation of T cell-mediated immune responses.     

EpCAM蛋白的表达、纯化及其单克隆抗体的制备

目的:原核表达EpCAM蛋白并制备抗EpCAM特异性单克隆抗体,初步鉴定相应单克隆抗体的特性。方法:PCR扩增EpCAM基因胞外区,将目的基因亚克隆至载体pET-28a(+),转化至大肠埃希菌株BL21,IPTG诱导表达,组氨酸亲和层析法纯化表达产物。纯化蛋白免疫BALB/c小鼠,将成功免疫的小鼠脾细胞与骨髓瘤SP2/0细胞融合,经ELISA筛选得到分泌特异性抗EpCAM的单克隆抗体的细胞株,免疫BALB/c小鼠进一步制备相应的单克隆抗体,并通过Western blot(蛋白质印记)和FACS(流式细胞分析)鉴定单抗的特异性及生物学活性。结果:成功构建重组表达载体pET28a-EpCAM并在大肠杆菌中获得表达,经His-tag亲和层析法获得纯化的EpCAM重组蛋白。EpCAM重组蛋白免疫的BALB/c小鼠的脾细胞与SP2/0细胞融合、筛选,获得两株稳定分泌EpCAM抗体的杂交瘤细胞株,分别命名为4B2、2F2并免疫BALB/c小鼠获得相应的单克隆抗体。Western blot结果显示4B2腹水纯化所得单抗能够识别FaDu细胞系(人咽鳞癌细胞)中的EpCAM蛋白,但2F2未能识别FaDu细胞中的变性的EpCAM蛋白。FACS结果显示两者均能和FaDu细胞中天然的EpCAM蛋白结合。讨论:成功制备了抗EpCAM的单克隆抗体,并能够识别人咽鳞癌细胞系FaDu中表达的EpCAM,为进一步研究EpCAM抗体在肿瘤治疗中的作用提供基础。     

Multifunction protein staphylococcal nuclease domain containing 1 (SND1) promotes tumor angiogenesis in human hepatocellular carcinoma through novel pathway that involves nuclear factor κB and miR-221

Staphylococcal nuclease domain-containing 1 (SND1) is a multifunctional protein that is overexpressed in multiple cancers, including hepatocellular carcinoma (HCC). Stable overexpression of SND1 in Hep3B cells expressing a low level of SND1 augments, whereas stable knockdown of SND1 in QGY-7703 cells expressing a high level of SND1 inhibits establishment of xenografts in nude mice, indicating that SND1 promotes an aggressive tumorigenic phenotype. In this study we analyzed the role of SND1 in regulating tumor angiogenesis, a hallmark of cancer. Conditioned medium from Hep3B-SND1 cells stably overexpressing SND1 augmented, whereas that from QGY-SND1si cells stably overexpressing SND1 siRNA significantly inhibited angiogenesis, as analyzed by a chicken chorioallantoic membrane assay and a human umbilical vein endothelial cell differentiation assay. We unraveled a linear pathway in which SND1-induced activation of NF-κB resulted in induction of miR-221 and subsequent induction of angiogenic factors Angiogenin and CXCL16. Inhibition of either of these components resulted in significant inhibition of SND1-induced angiogenesis, thus highlighting the importance of this molecular cascade in regulating SND1 function. Because SND1 regulates NF-κB and miR-221, two important determinants of HCC controlling the aggressive phenotype, SND1 inhibition might be an effective strategy to counteract this fatal malady.     

The Enhanced Metastatic Potential of Hepatocellular Carcinoma (HCC) Cells with Sorafenib Resistance

Acquired resistance towards sorafenib treatment was found in HCC patients, which results in poor prognosis. To investigate the enhanced metastatic potential of sorafenib resistance cells, sorafenib-resistant (SorR) cell lines were established by long-term exposure of the HCC cells to the maximum tolerated dose of sorafenib. Cell proliferation assay and qPCR of ABC transporter genes (ABCC1-3) were first performed to confirm the resistance of cells. Migration and invasion assays, and immunoblotting analysis on the expression of epithelial to mesenchymal transition (EMT) regulatory proteins were performed to study the metastatic potential of SorR cells. The expression of CD44 and CD133 were studied by flow cytometry and the gene expressions of pluripotency factors were studied by qPCR to demonstrate the enrichment of cancer stem cells (CSCs) in SorR cells. Control (CTL) and SorR cells were also injected orthotopically to the livers of NOD-SCID mice to investigate the development of lung metastasis. Increased expressions of ABCC1-3 were found in SorR cells. Enhanced migratory and invasive abilities of SorR cells were observed. The changes in expression of EMT regulatory proteins demonstrated an activation of the EMT process in SorR cells. Enriched proportion of CD44⁺ and CD44⁺CD133⁺ cells were also observed in SorR cells. All (8/8) mice injected with SorR cells demonstrated lung metastasis whereas only 1/8 mouse injected with CTL cells showed lung metastasis. HCC cells with sorafenib resistance demonstrated a higher metastatic potential, which may be due to the activated EMT process. Enriched CSCs were also demonstrated in the sorafenib resistant cells. This study suggests that advanced HCC patients with acquired sorafenib resistance may have enhanced tumor growth or distant metastasis, which raises the concern of long-term sorafenib treatment in advanced HCC patients who have developed resistance of sorafenib.     

Novel therapeutic strategies for targeting liver cancer stem cells

The cancer stem cell (CSC) hypothesis was first proposed over 40 years ago. Advances in CSC isolation were first achieved in hematological malignancies, with the first CSC demonstrated in acute myeloid leukemia. However, using similar strategies and technologies, and taking advantage of available surface markers, CSCs have been more recently demonstrated in a growing range of epithelial and other solid organ malignancies, suggesting that the majority of malignancies are dependent on such a compartment.Primary liver cancer consists predominantly of hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (ICC). It is believed that hepatic progenitor cells (HPCs) could be the origin of some HCCs and ICCs. Furthermore, stem cell activators such as Wnt/β-catenin, TGF-β, Notch and Hedgehog signaling pathways also expedite tumorigenesis, and these pathways could serve as molecular targets to assist in designing cancer prevention strategies. Recent studies indicate that additional factors such as EpCAM, Lin28 or miR-181 may also contribute to HCC progression by targeting HCC CSCs. Various therapeutic drugs that directly modulate CSCs have been examined in vivo and in vitro. However, CSCs clearly have a complex pathogenesis, with a considerable crosstalk and redundancy in signaling pathways, and hence targeting single molecules or pathways may have a limited benefit for treatment. Many of the key signaling molecules are shared by both CSCs and normal stem cells, which add further challenges for designing molecularly targeted strategies specific to CSCs but sparing normal stem cells to avoid side effects. In addition to the direct control of CSCs, many other factors that are needed for the maintenance of CSCs, such as angiogenesis, vasculogenesis, invasion and migration, hypoxia, immune evasion, multiple drug resistance, and radioresistance, should be taken into consideration when designing therapeutic strategies for HCC. Here we provide a brief review of molecular signaling in liver CSCs and present insights into new therapeutic strategies for targeting liver CSCs.     

Redundant and antagonistic functions of galectin-1, -3, and -8 in the elicitation of T cell responses

Galectins, a family of mammalian lectins, have emerged as key regulators of the immune response. We previously demonstrated that galectin (Gal)-8, from the tandem-repeat subgroup, exerts two well-defined effects on mouse naive peripheral CD4 T cells: Ag-specific costimulation and Ag-independent proliferation. These stimulatory signals on naive T cells have not been described for any other Gal. Therefore, we investigated whether Gal-1 and Gal-3, two prominent members of the Gal family, share the stimulatory effects exerted by Gal-8 on naive T cells. We found that Gal-1 costimulated Ag-specific T cell responses similarly to Gal-8, as evaluated in the DO11.10 TCR(OVA)-transgenic mouse model, by acting simultaneously on APCs and target CD4 T cells. In contrast, Gal-3 failed to costimulate Ag-specific T cell responses; moreover, it antagonized both Gal-1 and Gal-8 signals. We observed that both Gal-1 and Gal-3 were unable to induce Ag-independent proliferation; however, when two Gal-1 molecules were covalently fused, the resulting chimeric protein efficiently promoted proliferation. This finding indicates that Gal-1 might eventually induce proliferation and, moreover, stresses the requirement of a tandem-repeat structure. Remarkably, a single dose of recombinant Gal-1 or Gal-8 administered together with a suboptimal Ag dose to DO11.10 mice strengthened weak responses in vivo. Taken together, these findings argue for the participation of Gals in the initiation of the immune response and allow the postulation of these lectins as enhancers of borderline Ag responses, thus representing potential adjuvants for vaccine formulations.     

A quantitative proteomics analysis of MCF7 breast cancer stem and progenitor cell populations

Accumulating evidence has demonstrated that breast cancers are initiated and develop from a small population of stem‐like cells termed cancer stem cells (CSCs). These cells are hypothesized to mediate tumor metastasis and contribute to therapeutic resistance. However, the molecular regulatory networks responsible for maintaining CSCs in an undifferentiated state have yet to be elucidated. In this study, we used CSC markers to isolate pure breast CSCs fractions (ALDH+ and CD44+CD24‐ cell populations) and the mature luminal cells (CD49f‐EpCAM+) from the MCF7 cell line. Proteomic analysis was performed on these samples and a total of 3304 proteins were identified. A label‐free quantitative method was applied to analyze differentially expressed proteins. Using the criteria of greater than twofold changes and p value <0.05, 305, 322 and 98 proteins were identified as significantly different in three pairwise comparisons of ALDH+ versus CD44+CD24‐, ALDH+ versus CD49f‐EpCAM+ and CD44+CD24‐ versus CD49f‐EpCAM+, respectively. Pathway analysis of differentially expressed proteins by Ingenuity Pathway Analysis (IPA) revealed potential molecular regulatory networks that may regulate CSCs. Selected differential proteins were validated by Western blot assay and immunohistochemical staining. The use of proteomics analysis may increase our understanding of the underlying molecular mechanisms of breast CSCs. This may be of importance in the future development of anti‐CSC therapeutics.