CD44v6+ Hepatocellular Carcinoma Cells Maintain Stemness Properties through Met/cJun/Nanog Signaling.

Cancer stem cells (CSCs) are characterized by their self-renewal and differentiation abilities. CD44v6 is a novel CSC marker that can activate various signaling pathways. Here, we hypothesized that the HGF/Met signaling pathway promotes stemness properties in CD44v6+ hepatocellular carcinoma (HCC) cells via overexpression of the transcription factor, cJun, thus representing a valuable target for HCC therapy. Magnetic activated cell sorting was used to separate the CD44v6+ from CD44v6- cells, and Met levels were regulated using lentiviral particles and the selective Met inhibitor, PHA665752. An orthotopic liver xenograft tumor model was used to assess the self-renewal ability of CD44v6+ cells in immunodeficient NOD/SCID mice. Luciferase reporter and chromatin immunoprecipitation assays were also conducted using cJun-overexpressing 293 T cells to identify the exact binding site of cJun in the Nanog promoter. Our data demonstrate that CD44v6 is an ideal surface marker of liver CSCs. CD44v6+ HCC cells express higher levels of Met and possess self-renewal and tumor growth abilities. Xenograft liver tumors were smaller in nude mice injected with shMet HCC cells. Immunohistochemical analysis of liver tissue specimens revealed that high Met levels in HCC cells were associated with poor patient prognosis. Further, a cJun binding site was identified 1700 bp upstream of the Nanog transcription start site and mutation of the cJun binding site reduced Nanog expression. In conclusion, the HGF/Met signaling pathway is important for maintenance of stemness in CD44v6+ HCC cells by enhancing expression of cJun, which binds 1700 bp upstream of the Nanog transcription start site.

Internal Jugular Vein Thrombosis After Microwave Ablation of Cervical Lymph Node Metastasis in Papillary Thyroid Microcarcinoma: A Case Report.

In this study, two patients with papillary thyroid carcinoma and lymph node metastasis were treated by Dr. Shurong Wang's team and are reported. The two patients refused surgery and underwent microwave ablation (MWA) of the thyroid and lymph node lesions. Ultrasound review 2 days after MWA revealed internal jugular vein thrombosis. Patient #1 received low molecular weight heparin calcium injection, Xueshuantong injection, Xiangdan injection, and rivaroxaban. Patient #2 was treated with enoxaparin sodium injection, Xueshuantong injection, urokinase, and warfarin sodium tablet. The thrombus was successfully managed in each patient using anticoagulant treatment. Such complication of MWA has not been reported in many cases before. According to the relevant literature, thrombosis after thyroid cancer ablation might be related to subclinical hypothyroidism, increased heme oxidase 1 (HO-1) levels in the blood of patients with papillary thyroid cancer, and increased platelet content and mean platelet volume in patients with thyroid cancer. No specific cause of thrombosis was identified in the two cases reported here. No recurrence was observed after 1 (patient #1) and 4 (#2) years of follow-up. In conclusion, patients with papillary thyroid carcinoma and lymph node metastasis should undergo color Doppler ultrasound of the neck after MWA of thyroid lesions and neck metastasis.

Spindle and kinetochore-associated complex subunit 3 (SKA3) promotes stem cell-like properties of hepatocellular carcinoma cells through activating Notch signaling pathway.

BACKGROUND: Cancer stemness contributes to hepatocellular carcinoma (HCC) initiation, metastasis, drug resistance, and recurrence. The spindle and kinetochore-associated (SKA) complex has been shown to be involved in tumor progression; however, its effects on cancer stem cell-like properties have not yet been examined. This research sought to study each subunit of the SKA complex in HCC systematically. METHODS: Bioinformatic analyses were carried out to examine the expression and clinical data of the SKA complex's each subunit in HCC. The expression of the target genes was detected by quantitative reverse transcription-polymerase chain reaction and Western blot assays. Clone formation and Transwell assays were performed to assess the proliferation and migration abilities of the SKA complex's each subunit. Sphere formation assays and subcutaneous xenograft experiments were performed to investigate the effects of SKA complex subunit 3 (SKA3) on the self-renewal and tumorigenic abilities of HCC. RESULTS: Each subunit of the SKA complex was highly expressed in HCC, but only SKA complex subunit 1 (SKA1) and SKA3 were associated with the poor overall survival of HCC patients. Additionally, the HCC cells overexpressing SKA3 exhibited increased migration, invasion, proliferation, self-renewal, Sorafenib resistance and tumorigenic abilities. Notch signaling played a vital role in the process by which SKA3 promoted HCC stemness. CONCLUSIONS: SKA3 promotes HCC stem cell-like properties via the Notch signaling pathway. As SKA3 appears to act as a regulator of stemness in HCC, it might be a potential molecular target for HCC.

A Meta-Analysis on the Association Between TNFSF4 Polymorphisms (rs3861950 T > C and rs1234313 A > G) and Susceptibility to Coronary Artery Disease.

Background: Coronary artery disease (CAD) remains the leading cause of mortality worldwide, and its susceptibility is closely associated with genetic modifications. The association between inflammation and CAD has been investigated in detail. This meta-analysis was conducted based on the PRISMA guidelines to evaluate the association between the tumor necrosis factor superfamily member 4 (TNFSF4) gene polymorphisms (rs3861950 T > C and rs1234313 A > G) and the risk of CAD. Methods: The selected criteria included 11 eligible articles containing 18 studies (nine studies included 7,395 cases and 5,296 controls for rs3861950 and nine studies with 6,951 cases and 4,959 controls for rs1234313). Correlations between the two polymorphisms and CAD were estimated by pooling the odds ratios (ORs) with 95% confidence interval (95% CI) in allelic, dominant, recessive, heterozygous, and homozygous models. Results: The pooled analyses demonstrated that the rs3861950 T > C polymorphism was significantly associated with an increased risk of CAD in the Asian population in the allelic model, dominant model, and homozygous model. Furthermore, subgroup analysis based on disease type showed that TNFSF4 rs3861950 T > C had a robust correlation with increased risk of cerebral infarction (CI) in the allelic model, dominant model, heterozygous model, and homozygous model. However, the rs1234313 A > G polymorphism mostly tended to decrease the risk of CAD in the Asian and Caucasian populations in the allelic and dominant model. This single nucleotide polymorphism (SNP) had a close relation to myocardial infarction (MI) susceptibility in the allelic model, dominant model, and heterozygous model. Conclusion: This meta-analysis identified two novel SNPs in TNFSF4 significantly associated with CAD susceptibility.

US-guided Microwave Ablation of Low-Risk Papillary Thyroid Microcarcinoma: Longer-Term Results of a Prospective Study.

BACKGROUND: Papillary thyroid microcarcinoma (PTMC) has become a main cause of the extremely high incidence of thyroid carcinoma. This study aimed to evaluate the longer-term effectiveness of ultrasound (US)-guided microwave ablation (MWA) for treatment of low-risk PTMC with a large population. METHODS: This prospective study was approved by ethics committee of our institution. MWA was performed under US-guidance for 119 unifocal PTMC patients without clinically cervical or distant metastasis. The target ablation zone exceeded the tumor edge judged by contrast-enhanced US to avoid marginal residue and recurrence. US and thyroid function evaluation were followed at 1, 3, 6, and 12 months after treatment and every 6 to 12 months thereafter. Any adverse event associated with MWA was evaluated. RESULTS: The follow-up duration after MWA was 37.2 ± 20.9 months (range 12-101 months). Tumor volume decreased significantly from 1.87 ± 1.03 mL immediately after MWA to 0.01 ± 0.04 mL at the final evaluation (P < 0.001), with a mean volume reduction ratio of 99.4 ± 2.2% and 107 cases (93.9%) got complete remission. A patient was detected with cervical lymph node metastasis at 26-month follow-up and underwent 1 additional MWA treatment successfully. No distant metastasis was observed. All the acquired histological pathology results confirmed the absence of residual or recurrent tumor cells after MWA. No delayed complications associated with MWA were encountered for all patients. CONCLUSIONS: Percutaneous MWA is technically feasible for complete PTMC destruction and showed well longer-term effectiveness; thus, it seems to be an effective nonsurgical therapy to complement the current recommendation for selected low-risk PTMC patients.

Cancer-associated fibroblasts endow stem-like qualities to liver cancer cells by modulating autophagy.

PURPOSE: Both cancer-associated fibroblasts (CAFs) and liver cancer stem cells (LCSCs) play an important part in the tumorigenesis, development and metastasis of hepatocellular carcinoma (HCC). Moreover, the stem-like properties in HCC cells could be promoted by CAFs. However, the mechanism remains largely unknown. PATIENTS AND METHODS: We used conditioned medium (CM) of CAFs to culture Huh7 cells. Stemness of the cells was then examined mainly by sphere formation assay while stemness-associated genes including Nanog, Sox2 and Oct4 were measured by Western blotting. Immunofluorescence staining, Transmission Electron Microscope as well as Western blotting were performed to detect the level of autophagy in Huh7 cells. RESULTS: Increased level of stemness and autophagy was observed in HCC cells cultured in CAFs-CM compared to the control group. Activation of CAFs-induced autophagic flux could be inhibited by Chloroquine (CQ), which can accumulate LC3-II protein and increase punctate distribution of LC3 localization. Treatment of HCC cells with CQ effectively reversed the CAF-induced stemness, invasion, and metastasis ability in these cells. In vivo, Huh7 cells inoculated together with CAFs developed significantly larger tumors than Huh7 cells injected alone. Moreover, blockage of autophagy in Huh7 cells by CQ greatly reduced the growth of xenografted tumors of Huh7 cells combined with CAFs. CONCLUSION: These results reveal that CAFs are capable of promoting stemness and metastasis of HCC cells and blocking autophagy could markedly attenuate the stemness enhanced by CAFs, suggesting that targeting autophagy in HCC could be an effective strategy in HCC treatment.

Peri-tumor fibroblasts promote tumorigenesis and metastasis of hepatocellular carcinoma via Interleukin6/STAT3 signaling pathway.

Purpose: Because many hepatocellular carcinoma (HCC) cases develop from fibrotic or cirrhotic livers, fibroblasts are abundant in the microenvironment of HCC. Although the contribution of cancer-associated fibroblasts (CAFs) to the progression of HCC is well established, the role of fibroblasts has not been comprehensively revealed. Patients and methods: The RayBio Human Cytokine Antibody Array was used to elucidate the role of peri-tumor fibroblasts (PTFs) in promoting malignant properties of HCC. IL-6 and STAT3 signaling were inhibited in both HCC cell lines and non-tumor L-02 liver cells to further determine its role in the progression of HCC. Moreover, the expression of IL-6 and pTyr705 STAT3 was detected in HCC samples and peri-tumor liver tissues by immunohistochemical staining. Results: PTFs not only promoted the proliferation, invasion, and metastasis of liver cancer cells, but also stimulated the permanent malignant transformation of human non-tumor L-02 liver cells, resulting in hepatocarcinogenesis in vivo. The RayBio Human Cytokine Antibody Array indicated that PTFs secreted a higher level of soluble IL-6 than CAFs. IL-6 derived from PTFs greatly activated STAT3 Tyr705 phosphorylation in both non-tumor L-02 cells and HCC cells. IL-6-neutralizing antibody and STAT3 Tyr705 phosphorylation inhibitor, cryptotanshinone, largely abolished the positive effects of PTFs on HCC carcinogenesis and progression. Moreover, high expression of pTyr705 STAT3 in peri-tumor tissues was significantly correlated with tumor recurrence rate after three years in a postsurgical follow-up with patients with HCC. Conclusion: These results indicated that PTFs induce carcinogenesis and development of HCC via IL-6 and STAT3 signaling.

Musashi2 contributes to the maintenance of CD44v6+ liver cancer stem cells via notch1 signaling pathway.

BACKGROUND: Liver cancer stem cells (LCSCs) contribute to hepatocellular carcinoma (HCC) development, metastasis, and drug resistance. MSI2 and Notch1 signaling are involved in the maintenance of CSCs. However, it is unknown whether MSI2 and Notch1 are involved in the maintenance of CD44v6+ LCSCs. Therefore, we investigated the clinical significance and function of MSI2 and its relationship with Notch1 signaling in the maintenance of stemness properties in CD44v6+ LCSCs. METHODS: The expression of MSI2 and CD44v6 were detected by fresh specimens and a HCC tissue microarray. The tissue microarray containing 82 HCC samples was used to analyze the correlation between CD44v6 and MSI2. CD44v6+/- cells were isolated using microbeads sorting. We explored the roles of MSI2 and Notch1 signaling in CD44v6+ LCSCs by sphere formation assay, transwell assay, clone formation assay in vitro, and xenograft tumor models in vivo. A Notch RT2 PCR Array, Co-immunoprecipitation, and RNA-immunoprecipitation were used to further investigate the molecular mechanism of MSI2 in activating Notch1 signaling. RESULTS: Here, we found MSI2 expression was positively correlated with high CD44v6 expression in HCC tissues, and further correlated with tumor differentiation. CD44v6+ cells isolated from HCC cell lines exhibited increased self-renewal, proliferation, migration and invasion, resistance to Sorafenib and tumorigenic capacity. Both MSI2 and Notch1 signaling were elevated in sorted CD44v6+ cells than CD44v6- cells and played essential roles in the maintenance of stemness of CD44v6+ LCSCs. Mechanically, MSI2 directly bound to Lunatic fringe (LFNG) mRNA and protein, resulting in Notch1 activation. CONCLUSIONS: Our results demonstrated that MSI2 maintained the stemness of CD44v6+ LCSCs by activating Notch1 signaling through the interaction with LFNG, which could be a potential molecular target for stem cell-targeted therapy for liver cancer.

Cancer-associated fibroblasts promote the stemness of CD24+ liver cells via paracrine signaling.

Cancer stem cells (CSCs), which support tumor progress in hepatocellular carcinoma (HCC) developed in fibrotic or cirrhotic livers, are regulated by the tumor microenvironment. Cancer-associated fibroblasts (CAFs) are the major component of the tumor stroma in HCC; however, the mechanisms by which CAFs contribute to stemness maintenance remain largely unknown. Here, we found that the expression of CD24 was high in HCC tissues compared with adjacent normal liver tissues, and positively correlated with the poor prognosis and α-SMA expression in CAFs. CD24+ cells isolated from HCC cell lines exhibited stemness properties of self-renewal, chemotherapy resistance, metastasis, and tumorigenicity in NOD/SCID mice. Moreover, CAF-derived HGF and IL6 enhanced the stemness properties of CD24+ cells via activating STAT3 Tyr705 phosphorylation. Blockade of HGF/c-Met or IL6/IL6R signaling significantly abolished the effect of CAFs on stemness properties, which compromised the activation of STAT3 pathway in CD24+ cells. Meanwhile, knockdown of STAT3 in CD24+ cells notably attenuated CAF-induced stemness characteristics of CD24+ cells. Furthermore, in HCC patients, higher expression of phospho-STAT3 was also demonstrated to be positively correlated with poor clinical outcomes. In summary, HGF and IL6 secreted by CAFs promoted the stemness properties of CD24+ cells through the phosphorylation of STAT3 signaling, and targeting the paracrine pathways may provide a new therapeutic strategy for HCC. KEY MESSAGES: CD24, identified as a marker for HCC CSCs, was positively correlated with the poor prognosis and α-SMA expression in CAFs. CAFs promoted self-renewal, chemotherapy resistance, metastasis, and tumorigenicity of CD24+ HCC cells. HGF and IL6 secreted by CAFs promoted the stemness properties of CD24+ HCC cells through the phosphorylation of STAT3.

iNOS promotes CD24+CD133+ liver cancer stem cell phenotype through a TACE/ADAM17-dependent Notch signaling pathway.

The inducible nitric oxide synthase (iNOS) is associated with more aggressive solid tumors, including hepatocellular carcinoma (HCC). Notch signaling in cancer stem cells promotes cancer progression and requires Notch cleavage by ADAM (a disintegrin and metalloprotease) proteases. We hypothesized that iNOS/NO promotes Notch1 activation through TACE/ADAM17 activation in liver cancer stem cells (LCSCs), leading to a more aggressive cancer phenotype. Expression of the stem cell markers CD24 and CD133 in the tumors of patients with HCC was associated with greater iNOS expression and worse outcomes. The expression of iNOS in CD24+CD133+ LCSCs, but not CD24-CD133- LCSCs, promoted Notch1 signaling and stemness characteristics in vitro and in vivo, as well as accelerating HCC initiation and tumor formation in the mouse xenograft tumor model. iNOS/NO led to Notch1 signaling through a pathway involving the soluble guanylyl cyclase/cGMP/PKG-dependent activation of TACE/ADAM17 and up-regulation of iRhom2 in LCSCs. In patients with HCC, higher TACE/ADAM17 expression and Notch1 activation correlated with poor prognosis. These findings link iNOS to Notch1 signaling in CD24+CD133+ LCSCs through the activation of TACE/ADAM17 and identify a mechanism for how iNOS contributes to progression of CD24+CD133+ HCC.

Cancer-associated fibroblasts promote stem cell-like properties of hepatocellular carcinoma cells through IL-6/STAT3/Notch signaling.

In this study, we investigated the role of cancer-associated fibroblasts (CAFs) in hepatocellular carcinoma (HCC) progression. We showed that CAFs secrete high levels of IL-6, which promoted stem cell-like properties in HCC cells by activating Notch signaling through STAT3 Tyr705 phosphorylation. These effects were abolished by Notch1 shRNA knockdown in HCC cells or treatment with an IL-6 neutralizing antibody or the p-STAT3 (Tyr705) inhibitor cryptotanshinone. Xenografted liver tumors were larger in nude mice injected with HCC cells and CAFs than in those receiving HCC cells alone. Moreover, immunohistochemical analysis of liver tissue specimens from 88 HCC patients revealed that high nuclear Notch intracellular domain (nNICD) levels in HCC cells correlated with poor prognosis in patients. These findings suggest that IL-6 secreted by CAFs promotes stem cell-like properties in HCC cells by enhancing STAT3/Notch signaling.

Ultrasound guided percutaneous microwave ablation of benign thyroid nodules: safety and imaging follow-up in 222 patients.

OBJECTIVE: Microwave ablation is a minimally invasive technique that has been used to treat benign and malignant tumors of liver, lung and kidney. Towards thyroid nodules, only a few cases are reported so far. The aim of the study was to investigate the effectiveness and safety of ultrasound-guided percutaneous microwave ablation in the treatment of benign thyroid nodules with a large sample. MATERIALS AND METHODS: A total of 477 benign thyroid nodules in 222 patients underwent microwave ablation in our department from July 2009 to March 2012. Microwave ablation was carried out using microwave antenna (16G) under local anesthesia. Nodule volume, thyroid function and clinical symptoms were evaluated before treatment and at 1, 3, more than 6 months. The study was ethics committee approved and written informed consents were obtained from all patients. RESULTS: All thyroid nodules significantly decreased in size after microwave ablation. A 6-month follow-up was achieved in 254 of 477 nodules, and the mean decrease in the volume of thyroid nodules was from 2.13 ± 4.42 ml to 0.45 ± 0.90 ml, with a mean percent decrease of 0.65 ± 0.65. A volume-reduction ratio greater than 50% was observed in 82.3% (209/254) of index nodules, and 30.7% (78/254) of index nodules disappeared 6-month after the ablation. The treatment was well tolerated and no major complications were observed except pain and transient voice changes. CONCLUSIONS: Microwave ablation seems to be a safe and effective technique for the treatment of benign thyroid nodules. Further prospective randomized studies are needed to define the role of the procedure in the treatment of thyroid nodules.