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Supplementary Figure 1. Validation of the lncRNA microarray data using qRT-PCR. Supplementary Figure 2. Expression levels of DZnep-induced lncRNAs in a panel of HCC cell lines and non-tumor cell lines. Supplementary Figure 3. Expression levels of DZnep-induced lncRNAs in paired human HCC tissues and adjacent non-tumor tissues. Supplementary Figure 4. High specificity of ChIP assay in measuring true interaction between EZH2 and the promoters of EZH2 targeted genes. Supplementary Figure 5. Inhibition of TCAM1P-004 and RP11-598D14.1 with siRNAs promoted cell proliferation. Supplementary Figure 6. Knockdown of TCAM1P-004 and RP11-598D14.1 with shRNAs inhibited cell apoptosis, promoted colony formation ability and transformation ability, and stimulated tumor growth in xenograft mice model. Supplementary Figure 7. Overexpression of TCAM1P-004 and RP11-598D14.1 repressed cell colony formation and transformation abilities, and tumor growth in xenograft mice model. Supplementary Figure 8. Overexpression of TCAM1P-004 or RP11-598D14.1 promoted apoptosis in subcutaneous tumors derived from Hep3B cells. Supplementary Figure 9. Inhibition of TCAM1P-004 or RP11-598D14.1 could induce tumorigenesis of non-tumor MIHA cells. Supplementary Figure 10. KEGG pathway enrichment analysis of TCAM1P-004 and RP11-598D14.1 regulated genes. Supplementary Figure 11. qRT-PCR validated signaling pathways related to TCAM1P-004 and RP11-598D14.1. Supplementary Figure 12. Expression of DDIT and PFKFB4 in HCC cell lines and HCC tumor samples. Supplementary Figure 13. Expression of PFKFB4 in HCC tumor samples. Supplementary Figure 14. Alignment analysis between lncRNA TCAM1P-004 and RP11-598D14.1. Supplementary Figure 15. EZH1 showed no significant role in HCC.

Figure S1. Dynamic change of the clustering status of circulating tumor cells (CTCs) along their circulating route. Figure S2. Variation of circulating tumor cell (CTC) clustering status between primary hepatocellular carcinoma (HCC) efferent and afferent micovessels. Example of circulating tumor microemboli (CTM) detected in microscopic hepatic vein (mHV, efferent vessel) and singular CTCs in microscopic hepatic artery (mHA, afferent vessel) stained for epithelial (E) and mesenchymal (M) markers. E-cad = E-cadherin, red; CK = cytokeratin, brown; vit = vimentin, brown. Scale bar: original magnification, 100μm; inserts, 25 μm. Figure S3. Determination of epithelial-mesenchymal transition (EMT)-related marker expression on hepatocellular carcinoma (HCC) cell lines. Figure S4. Circulating tumor cells (CTCs) isolated using the CELLSEARCH® system were verified by confocal microscopy. Figure S5. Scatter plots showing the comparison of the cell counts of each EMT-related CTC phenotype from five vascular sites, including HV, PA, PV, IHIVC, and PoV (*P

Purpose: The spatial heterogeneity of phenotypic and molecular characteristics of CTCs within the circulatory system remains unclear. Herein, we mapped the distribution and characterized biological features of CTCs along the transportation route in hepatocellular carcinoma (HCC).Experimental Design: In 73 localized HCC patients, blood was drawn from peripheral vein (PV), peripheral artery (PA), hepatic veins (HV), infrahepatic inferior vena cava (IHIVC), and portal vein (PoV) before tumor resection. Epithelial and mesenchymal transition (EMT) phenotype in CTCs were analyzed by a 4-channel immunofluorescence CellSearch assay and microfluidic quantitative RT-PCR. The clinical significance of CTCs from different vascular sites was evaluated.Results: The CTC number and size gradient between tumor efferent vessels and postpulmonary peripheral vessels was marked. Tracking the fate of CTC clusters revealed that CTCs displayed an aggregated–singular-aggregated manner of spreading. Single-cell characterization demonstrated that EMT status of CTCs was heterogeneous across different vascular compartments. CTCs were predominantly epithelial at release, but switched to EMT-activated phenotype during hematogeneous transit via Smad2 and β-catenin related signaling pathways. EMT activation in primary tumor correlated with total CTC number at HV, rather than epithelial or EMT-activated subsets of CTCs. Follow-up analysis suggested that CTC and circulating tumor microemboli burden in hepatic veins and peripheral circulation prognosticated postoperative lung metastasis and intrahepatic recurrence, respectively.Conclusions: The current data suggested that a profound spatial heterogeneity in cellular distribution and biological features existed among CTCs during circulation. Multivascular measurement of CTCs could help to reveal novel mechanisms of metastasis and facilitate prediction of postoperative relapse or metastasis pattern in HCC. Clin Cancer Res; 24(3); 547–59. ©2017 AACR.

Long noncoding RNAs (lncRNA) play critical roles in the development of cancer, including hepatocellular carcinoma (HCC). However, the mechanisms underlying their deregulation remain largely unexplored. In this study, we report that two lncRNAs frequently downregulated in HCC function as tumor suppressors and are epigenetically silenced by histone methyltransferase EZH2. lncRNAs TCAM1P-004 and RP11-598D14.1 were inhibited by EZH-mediated trimethylation of H3K27me3 at their promoters. Downregulation of TCAM1P-004 and RP11-598D14.1 was frequently observed in HCC tumors compared with adjacent normal tissues. Both lncRNAs inhibited cell growth, cell survival, and transformation in HCC cells in vitro as well as tumor formation in vivo. Using RNA pull-down and mass spectrometry, we demonstrated that TCAM1P-004 bound IGF2BP1 and HIST1H1C, whereas RP11-598D14.1 bound IGF2BP1 and STAU1. These lncRNA–protein interactions were critical in regulating p53, MAPK, and HIF1α pathways that promoted cell proliferation in HCC. Overexpression of EZH2 was critical in repressing TCAM1P-004 and RP11-598D14.1, and EZH2-TCAM1P-004/RP11-598D14.1–regulated pathways were prevalent in human HCC. Aberrant suppression of TCAM1P-004 and RP11-598D14.1 led to loss of their tumor-suppressive effects by disrupting the interaction with IGF2BP1, HIST1H1C, and STAU1, which in turn promoted HCC development and progression. Collectively, these findings demonstrate the role of TCAMP1P-004 and RP11-598D14.1 in suppressing tumor growth and suggest that EZH2 may serve as a therapeutic target in HCC.Significance:EZH2-mediated loss of lncRNAs TCAM1P-004 and RP11-598D14.1 hinders the formation of tumor suppressor lncRNA–protein complexes and subsequently promotes HCC growth.

Although in the last few decades we have witnessed the rapid development of treatments for non-small cell lung cancer (NSCLC), it still remains the leading cause of cancer-related death. Increasing efforts have been devoted to exploring potential biomarkers and molecular targets for NSCLC. Foxp3, a transcription factor that was discovered as a master regulator of regulatory T cells (Tregs), has been found to express abnormally in tumoral cells including lung cancer cells. In recent years, increasing evidence have surfaced, revealing the carcinogenic effect of FOXP3 in lung cancer. In this review, we analyzed and summarized the function of FOXP3, its regulation and therapeutic potentials in NSCLC, with a hope to facilitate the development of novel treatments for NSCLC.

Transcription factor FOXP3 is a crucial regulator in the development and function of regulatory T cells (Treg) that are essential for immunological tolerance and homeostasis. Numerous studies have indicated the correlation of tumor infiltrating FOXP3+ Treg upregulation with poor prognostic parameters in thyroid cancer, including lymph node metastases, extrathyroidal extension, and multifocality. Most immune-checkpoint molecules are expressed in Treg. The blockage of such signals with checkpoint inhibitors has been approved for several solid tumors, but not yet for thyroid cancer. Thyroid abnormalities may be induced by checkpoint inhibitors. For example, hypothyroidism, thyrotoxicosis, painless thyroiditis, or even thyroid storm are more frequently associated with anti-PD-1 antibodies (pembrolizumab and nivolumab). Therefore, Targeting FOXP3+ Treg may have impacts on checkpoint molecules and the growth of thyroid cancer. Several factors may impact the role and stability of FOXP3, such as alternative RNA splicing, mutations, and post-translational modification. In addition, the role of FOXP3+ Treg in the tumor microenvironment is also affected by the complex regulatory network formed by FOXP3 and its transcriptional partners. Here we discussed how the expression and function of FOXP3 were regulated and how FOXP3 interacted with its targets in Treg, aiming to help the development of FOXP3 as a potential therapeutic target for thyroid cancer.

Multiportal video-assisted thoracic surgery (VATS) for major lung resection causes less immunochemokine production compared to thoracotomy. Whether uniportal VATS is similarly associated with lower early postoperative circulating levels of immunochemokines compared to multiportal VATS have not been studied. Selected patients who received uniportal or multiportal VATS major lung resection were recruited. Blood samples were collected preoperatively and on postoperative days 1 and 3 for enzyme linked immunosorbent assay of serum levels of Tissue Inhibitor of Metalloproteinase (TIMP)-1, Insulin Growth Factor Binding Protein (IGFBP)-3, and Matrix Metalloproteinase (MMP)-9. A linear mixed-effects models were used to analyze the effects of uniportal VATS on the postoperative circulating chemokine levels. From March 2014 to April 2017, 68 consecutive patients consented for the prospective study and received major lung resection by either uniportal VATS (N = 29) or multiportal VATS (N = 39) were identified. Uniportal VATS major lung resection was associated with lower post-operative levels of TIMP-1 and MMP-9 compared to multiportal VATS after controlling for the effects of the corresponding baseline level and the time of follow-up measurement. No difference was observed for the level of IGFBP-3. Less immunochemokine disturbances was observed after uniportal VATS major lung resection compared to multiportal VATS.

Rationale: Hyperactivation of HGF/MET signaling pathway is a critical driver in liver tumorigenesis. Cytochrome P450 1A2 (CYP1A2) was significantly down-regulated in hepatocellular carcinoma (HCC). However, little is explored about its tumor suppressive role in HCC. In this study, we examined the functional mechanisms and clinical implication of CYP1A2 in HCC. Methods: The clinical impact of CYP1A2 was evaluated in HCC patients in Hong Kong cohort. The biological functions of CYP1A2 were investigated in vitro and in vivo. A series of biochemical experiments including Western blot assay, immunohistochemistry, quantitative reverse transcription-polymerase chain reaction, and Co-immunoprecipitation assay were conducted. Results: CYP1A2 expression was prominently silenced in HCC tumor tissues and the high expression of CYP1A2 was significantly correlated with lower AFP level, less vascular invasion, and better tumor-free survival in local cohort of HCC patients. The overexpression of CYP1A2 inhibited HCC cell viability and clonogenicity, reduced cell migration and invasion abilities in vitro, and suppressed tumorigenicity in vivo, whereas CYP1A2 knockdown exhibited the opposite effects. CYP1A2 significantly hindered HGF/MET signaling and Matrix metalloproteinases (MMPs) expression in HCC cells. Mechanically, CYP1A2 decreased HGF level and diminished HIF-1α expression, both of which are recognized as key regulators of MET activation. As the transcriptional activator of MET, HIF-1α was identified as a binding partner of CYP1A2. Direct binding of CYP1A2 with HIF-1α induced ubiquitin-mediated degradation of HIF-1α, inhibiting HIF-1α-mediated transcriptions. Conclusions: In conclusion, our results have identified CYP1A2 as a novel antagonist of HGF/MET signaling, and CYP1A2 may serve as an independent new biomarker for the prognosis of HCC patients.

Hepatocellular carcinoma (HCC) is a leading cause of cancer mortality worldwide. The outcome of current standard treatments, as well as targeted therapies in advanced stages, are still unsatisfactory. Attention has been drawn to novel strategies for better treatment efficacy. Hepatocyte growth factor/c-mesenchymal-epithelial transition factor (HGF/c-Met) axis has been known as an essential element in the regulation of liver diseases and as an oncogenic factor in HCC. In this review, we collected the evidence of HGF/c-Met as a tumor progression and prognostic marker, discussed the anti-c-Met therapy in vitro, summarized the outcome of c-Met inhibitors in clinical trials, and identified potential impetus for future anti-c-Met treatments. We also analyzed the inconsistency of HGF/c-Met from various publications and offered reasonable explanations based on the current understanding in this area. In conclusion, HGF/c-Met plays a crucial role in the progression and growth of HCC, and the strategies to inhibit this pathway may facilitate the development of new and effective treatments for HCC patients.

The transcription factor forkhead box P3 (FOXP3) is a biomarker for regulatory T cells and can also be expressed in cancer cells, but its function in cancer appears to be divergent. The role of hepatocyte-expressed FOXP3 in hepatocellular carcinoma (HCC) is unknown. Here, we collected tumor samples and clinical information from 115 HCC patients and used five human cancer cell lines. We examined FOXP3 mRNA sequences for mutations, used a luciferase assay to assess promoter activities of FOXP3's target genes, and employed mouse tumor models to confirm in vitro results. We detected mutations in the FKH domain of FOXP3 mRNAs in 33% of the HCC tumor tissues, but in none of the adjacent nontumor tissues. None of the mutations occurred at high frequency, indicating that they occurred randomly. Notably, the mutations were not detected in the corresponding regions of FOXP3 genomic DNA, and many of them resulted in amino acid substitutions in the FKH region, altering FOXP3's subcellular localization. FOXP3 delocalization from the nucleus to the cytoplasm caused loss of transcriptional regulation of its target genes, inactivated its tumor-inhibitory capability, and changed cellular responses to histone deacetylase (HDAC) inhibitors. More complex FKH mutations appeared to be associated with worse prognosis in HCC patients. We conclude that mutations in the FKH domain of FOXP3 mRNA frequently occur in HCC and that these mutations are caused by errors in transcription and are not derived from genomic DNA mutations. Our results suggest that transcriptional mutagenesis of FOXP3 plays a role in HCC.

Liver cancer stem cells (LCSCs) initiate hepatocellular carcinoma (HCC) and contribute to its recurrence and treatment resistance. Studies have suggested ZBP-89 as a candidate tumor suppressor in HCC. We explored the role of ZBP-89 in the regulation of LCSCs. This study was performed in liver tissue samples from 104 HCC patients, 2 cell lines and mouse tumor models. We demonstrated that ZBP-89 was weakly expressed in LCSCs. Patients with high expression of LCSC markers displayed reduced survivals and higher recurrence rates after curative surgical operation. The expression of ZBP-89 was predictive for decreased recurrence. LCSC markers were negatively correlated with ZBP-89 in HCC tissues and in enriched liver tumor spheres. The exogenous expression of ZBP-89 attenuated the tumor-sphere formation and secondary colony formation capabilities of LCSCs in vitro and tumorigenicity in vivo. Furthermore, the negative effect of ZBP-89 on cancer stemness was Notch1-dependent. Localized with Notch1 intracellular domain (NICD1) in the nucleus, ZBP-89 repressed the Notch1 signaling pathway by competitive binding to NICD1 with MAML1. Collectively, ZBP-89 negatively regulates HCC stemness via inhibiting the Notch1 signaling.

PurposeThe inhibition of estrogen receptor alpha (ERα) or the activation of ERβ can inhibit papillary thyroid cancer (PTC), but the precise mechanism is not known. We aimed to explore the role of ERα and ERβ on the production of endogenous peroxisome proliferator-activated receptor gamma (PPARγ) ligands in PTC.Methods2 PTC cell lines, 32 pairs of PTC tissues and matched normal thyroid tissues were used in this study. The levels of endogenous PPARγ ligands 15(S)-hydroxyeicosatetraenoic acid (15(S)-HETE), 13-S-hydroxyoctadecadienoic acid (13(S)-HODE), and15-deoxy-Δ12,14-prostaglandin J2 (PGJ2) were measured by ELISA.ResultsThe levels of PGJ2 and 15(S)-HETE were significantly reduced in PTC, but 13(S)-HODE was not changed. Activation of ERα or inhibition of ERβ significantly downregulated the production of PGJ2, 15(S)-HETE and 13(S)-HODE, whereas inhibition of ERα or activation of ERβ markedly upregulated the production of these three ligands. Application of endogenous PPARγ ligands inhibited growth, induced apoptosis of cancer cells, and promoted the efficacy of chemotherapy.ConclusionThe levels of endogenous PPARγ ligands PGJ2 and 15(S)-HETE are significantly decreased in PTC. The inhibition of ERα or activation of ERβ can inhibit PTC by stimulating the production of endogenous PPARγ ligands to induce apoptosis in cancer cells.

Long noncoding RNAs (lncRNA) play critical roles in the development of cancer, including hepatocellular carcinoma (HCC). However, the mechanisms underlying their deregulation remain largely unexplored. In this study, we report that two lncRNAs frequently downregulated in HCC function as tumor suppressors and are epigenetically silenced by histone methyltransferase EZH2. lncRNAs TCAM1P-004 and RP11-598D14.1 were inhibited by EZH-mediated trimethylation of H3K27me3 at their promoters. Downregulation of TCAM1P-004 and RP11-598D14.1 was frequently observed in HCC tumors compared with adjacent normal tissues. Both lncRNAs inhibited cell growth, cell survival, and transformation in HCC cells in vitro as well as tumor formation in vivo. Using RNA pull-down and mass spectrometry, we demonstrated that TCAM1P-004 bound IGF2BP1 and HIST1H1C, whereas RP11-598D14.1 bound IGF2BP1 and STAU1. These lncRNA–protein interactions were critical in regulating p53, MAPK, and HIF1α pathways that promoted cell proliferation in HCC. Overexpression of EZH2 was critical in repressing TCAM1P-004 and RP11-598D14.1, and EZH2-TCAM1P-004/RP11-598D14.1–regulated pathways were prevalent in human HCC. Aberrant suppression of TCAM1P-004 and RP11-598D14.1 led to loss of their tumor-suppressive effects by disrupting the interaction with IGF2BP1, HIST1H1C, and STAU1, which in turn promoted HCC development and progression. Collectively, these findings demonstrate the role of TCAMP1P-004 and RP11-598D14.1 in suppressing tumor growth and suggest that EZH2 may serve as a therapeutic target in HCC. Significance: EZH2-mediated loss of lncRNAs TCAM1P-004 and RP11-598D14.1 hinders the formation of tumor suppressor lncRNA–protein complexes and subsequently promotes HCC growth.

Background Cultured human cells are pivotal models to study human gene functions, but introducing complete loss of function in diploid or aneuploid cells has been a challenge. The recently developed CRISPR/Cas9-mediated homology-independent knock-in approach permits targeted insertion of large DNA at high efficiency, providing a tool for insertional disruption of a selected gene. Pioneer studies have showed promising results, but the current methodology is still suboptimal and functional outcomes have not been well examined. Taking advantage of the promoterless fluorescence reporter systems established in our previous study, here, we further investigated potentials of this new insertional gene disruption approach and examined its functional outcomes. Results Exemplified by using hyperploid LO2 cells, we demonstrated that simultaneous knock-in of dual fluorescence reporters through CRISPR/Cas9-induced homology-independent DNA repair permitted one-step generation of cells carrying complete disruption of target genes at multiple alleles. Through knocking-in at coding exons, we generated stable single-cell clones carrying complete disruption of ULK1 gene at all four alleles, lacking intact FAT10 in all three alleles, or devoid of intact CtIP at both alleles. We have confirmed the depletion of ULK1 and FAT10 transcripts as well as corresponding proteins in the obtained cell clones. Moreover, consistent with previous reports, we observed impaired mitophagy in ULK1−/− cells and attenuated cytokine-induced cell death in FAT10−/− clones. However, our analysis showed that single-cell clones carrying complete disruption of CtIP gene at both alleles preserved in-frame aberrant CtIP transcripts and produced proteins. Strikingly, the CtIP-disrupted clones raised through another two distinct targeting strategies also produced varied but in-frame aberrant CtIP transcripts. Sequencing analysis suggested that diverse DNA processing and alternative RNA splicing were involved in generating these in-frame aberrant CtIP transcripts, and some infrequent events were biasedly enriched among the CtIP-disrupted cell clones. Conclusion Multiallelic gene disruption could be readily introduced through CRISPR/Cas9-induced homology-independent knock-in of dual fluorescence reporters followed by direct tracing and cell isolation. Robust cellular mechanisms exist to spare essential genes from loss-of-function modifications, by generating partially functional transcripts through diverse DNA and RNA processing mechanisms. Electronic supplementary material The online version of this article (10.1186/s12915-018-0616-2) contains supplementary material, which is available to authorized users.

Papillary thyroid cancer can dedifferentiate into a much more aggressive form of thyroid cancer, namely into anaplastic thyroid cancer. Nrf2 is commonly activated in papillary thyroid cancer, whereas its role in anaplastic thyroid cancer has not been fully explored. In this study, we used two cell lines and an animal model to examine the function of Nrf2 in anaplastic thyroid cancer. The role of Nrf2 in anaplastic thyroid cancer was investigated by a series of functional studies in two anaplastic thyroid cancer cell lines, FRO and KAT-18, and further confirmed with an in vivo study. The impact of Nrf2 on the sensitivity of anaplastic thyroid cancer cells to lenvatinib was also investigated to evaluate its potential clinical implication. We found that the expression of Nrf2 was significantly higher in anaplastic thyroid cancer cell line cells than in papillary thyroid cancer cells or normal control cells. Knockdown of Nrf2 in anaplastic thyroid cancer cells inhibited their viability and clonogenicity, reduced their migration and invasion ability in vitro, and suppressed their tumorigenicity in vivo. Mechanistically, knockdown of Nrf2 decreased the expression of Notch1. Lastly, knockdown of Nrf2 increased the sensitivity of anaplastic thyroid cancer cells to lenvatinib. As knockdown of Nrf2 reduced the metastatic and invasive ability of anaplastic thyroid cancer cells by inhibiting the Notch 1 signaling pathway and increased the cancer cell sensitivity to lenvatinib, Nrf2 could be a promising therapeutic target for patients with anaplastic thyroid cancer.

The status of FOXP3 and its isoforms in hepatocellular carcinoma (HCC) is unclear. We aimed to investigate the expression and function of FOXP3 and its isoforms in HCC. The study was performed on 84 HCC patients, HCC cell lines and a mouse tumor model. The levels of FOXP3 and its isoforms were determined by nested PCR, quantitative real-time PCR and immunohistochemistry (IHC) staining. The correlation between their levels and clinicopathologic characteristics was analyzed. The full length of FOXP3 (FOXP3) and exon 3-deleted FOXP3 (FOXP3Δ3) were found to be the major isoforms in HCC. The levels of FOXP3Δ3 mRNA and protein in HCC tumor samples were not significantly different from their adjacent normal tissues. The high expression of FOXP3 protein in HCC patients showed a good overall survival. The overexpression of FOXP3 significantly reduced tumor cell proliferation, migration and invasion. The immunofluorescence result indicated that FOXP3 needed to be translocated into the nucleus to exert its inhibitory function. The luciferase assay demonstrated that FOXP3 could be synergistic with Smad2/3/4 to inhibit the oncogene c-Myc. The co-immunoprecipitation results further revealed that FOXP3 could interact with Smad2/3/4. The chromatin immunoprecipitation (ChIP) assay showed that both FOXP3 and Smad2/3/4 bound the promoter of the c-Myc to inhibit it. The in vivo mouse tumor model study confirmed the inhibitory effect of FOXP3. Collectively, the expression of tumor FOXP3 can inhibit the growth of HCC via suppressing c-Myc directly or indirectly via interacting with Smad2/3/4. Therefore, FOXP3 is a tumor suppressor in HCC.

Cisplatin is a chemotherapy drug that has been used to treat a number of cancers for decades, and is still one of the most commonly used anti-cancer agents. However, some patients do not respond to cisplatin while other patients who were originally sensitive to cisplatin eventually develop chemoresistance, leading to treatment failure or/and tumor recurrence.Different mechanisms contribute to cisplatin resistance or sensitivity, involving multiple pathways or/and processes such as DNA repair, DNA damage response, drug transport, and apoptosis. Among the various mechanisms, it appears that microRNAs play an important role in determining the resistance or sensitivity. In this article, we analyzed and summarized recent findings in this area, with the aim that these data can aid further research and understanding, leading to the eventual reduction of cisplatin resistance.microRNAs can positively or negatively regulate cisplatin resistance by acting on molecules or/and pathways related to apoptosis, autophagy, hypoxia, cancer stem cells, NF-κB, and Notch1. It appears that the modulation of relevant microRNAs can effectively re-sensitize cancer cells to cisplatin regimen in certain types of cancers including breast, colorectal, gastric, liver, lung, ovarian, prostate, testicular, and thyroid cancers.

Normal thyroid tissue displays a prevalent expression of ERβ than ERα, which drastically turns upside down in the initiation and progression of papillary thyroid cancer (PTC). The underlying molecular mechanism of this phenomenon remains unclear. Here, we demonstrated that ERα and ERβ were coexpressed in human thyroid tissues and cells. ERα mRNA (A-1) and ERβ mRNA (0N-1), transcribed from Promoter A of ERα gene and Promoter 0N of ERβ gene, respectively, were the major mRNA isoforms which mainly contributed to total ERα mRNA and total ERβ mRNA in human thyroid-derived cell lines and tissues. The expression levels of ERα mRNA (A-1) and total ERα mRNA were gradually increased, and those of ERβ mRNA (0N-1) and total ERβ mRNA were decreased by degree in the initiation and progression of PTC. No aberrant DNA methylation of ERα 5'-untranslated region was involved in its up-regulation; however, aberrant DNA methylation in Promoter 0N and Exon 0N of ERβ gene was found to be involved in its down-regulation in the initiation and progression of PTC. ERβ can repress ERα gene transcription via recruitment of NCoR and displacement of RNA polymerase II at the Sp1 site in ERα Promoter A-specific region in thyroid-derived cells. It is suggested that DNA methylation of CpG islands in Promoter 0N and Exon 0N of ERβ gene leads to a decreased ERβ gene expression, which attenuates its inhibitory effect on ERα gene transcription and results in an increased ERα gene expression, cell proliferation, initiation, and progression of PTC.

Metformin, an anti-hyperglycemic drug, has been known to have antitumor properties for around 15 years. Although there are a number of reports attributing the antitumor function of metformin to its impact on energy homeostasis and oxygen re-distribution in tumor microenvironment, detailed mechanisms remain largely unknown. In the past several years, there is an increasing number of publications indicating that metformin can affect various immunological components including lymphocytes, macrophages, cytokines and several key immunological molecules in both human and animal studies. These interesting results appear to be in line with emerging data that suggest associations between immune responses and energy homeostasis/oxygen re-distribution, which may explain effective impacts of metformin on immunotherapies against autoimmune diseases as well as cancers. This review article is to analyse and discuss recent development in the above areas with aim to justify metformin as a new adjuvant for immunotherapy against human cancers. We hope that our summary will help to optimize the application of metformin for various types of human cancers.

Sorafenib resistance has become the main obstacle in the effective treatment of advanced hepatocellular carcinoma (HCC) patients. Activation of nuclear factor kappa B (NF-κB) is a newly identified mechanism that contributes to desensitized sorafenib. Cytochrome P450 1A2 (CYP1A2) functions as a tumor suppressor in HCC and its expression is negatively associated with NF-κB in the liver. This study aimed to study whether CYP1A2 could overcome sorafenib resistance. To investigate whether CYP1A2 and NF-κB p65 played roles in sorafenib desensitization, we established sorafenib-resistant (SR) HCC cells. SR cells decreased the expression of CYP1A2 along with the upregulation of NF-κB p65. CYP1A2 overexpression attenuated SR cell proliferation, increased sorafenib sensitivity, and inhibited the NF-κB pathway, whereas CYP1A2 silence showed opposite effects. Sorafenib, in combination with omeprazole, a CYP1A2 inducer, significantly hindered the growth and invasion of SR cells in vitro as well as decreased the tumor growth in vivo. The combination treatment markedly increased CYP1A2 expression and inhibited the sorafenib-induced NF-κB signaling. In addition, the overexpression of NF-κB p65 stimulated the SR cell growth and desensitized sorafenib in SR cells, where CYP1A2 overexpression reversed the phenomenon. Lastly, the majority of HCC tissue samples displayed decreased CYP1A2 but increased NF-κB p65 protein expression. Collectively, CYP1A2 can sensitize SR cells to sorafenib via inhibiting NF-κB p65 axis. Omeprazole in combination with sorafenib exerts a synergistic effect in alleviating acquired sorafenib resistance.

Background Translationally controlled tumor protein (TCTP) is linked to lung cancer. However, upon lung cancer carcinogens stimulation, there were no reports on the relationship between TCTP and lung cell carcinogenic epithelial–mesenchymal transition (EMT). This study was designed to investigate the molecular mechanism of regulation of TCTP expression and its role in lung carcinogens-induced EMT. Methods To study the role of TCTP in lung carcinogens [particulate matter 2.5 (PM2.5) or 4-methylnitrosamino-l-3-pyridyl-butanone (NNK)]-induced EMT, PM2.5/NNK-treated lung epithelial and non-small cell lung cancer (NSCLC) cells were tested. Cell derived xenografts, human lung cancer samples and online survival analysis were used to confirm the results. MassArray assay, Real-time PCR and Reporter assays were performed to elucidate the mechanism of regulation of TCTP expression. All statistical analyses were performed using GraphPad Prism version 6.0 or SPSS version 20.0. Results Translationally controlled tumor protein and vimentin expression were up-regulated in PM2.5/NNK-treated lung cells and orthotopic implantation tumors. TCTP expression was positively correlated with vimentin in human NSCLC samples. Patients with high expression of TCTP displayed reduced overall and disease-free survival. TCTP overexpression could increase vimentin expression and promote cell metastasis. Furthermore, PM2.5/NNK stimulation brought a synergistic effect on EMT in TCTP-transfected cells. TCTP knockdown blocked PM2.5/NNK carcinogenic effect. Mechanically, PM2.5/NNK-induced TCTP expression was regulated by one microRNA, namely miR-125a-3p, but not by methylation on TCTP gene promoter. The level of TCTP was regulated by its specific microRNA during the process of PM2.5/NNK stimulation, which in turn enhanced vimentin expression and played a permissive role in carcinogenic EMT. Conclusions Our results provided new insights into the mechanisms of TCTP regulatory expression in lung carcinogens-induced EMT. TCTP and miR-125a-3p might act as potential prognostic biomarkers and therapeutic targets for NSCLC.

INTRODUCTION Zinc-binding protein-89 (ZBP-89) is a Kruppel-type zinc-finger transcription factor that regulates target gene expression profiles via directly binding to GC-rich gene promoters, recruiting chromatin modifiers or by interacting with other proteins. The importance of ZBP-89 in the regulation of cell cycle arrest and apoptosis has led to increased interest and investigations for its role in cancer development. Areas covered: We describe ZBP-89 as a candidate therapeutic target for hepatocellular carcinoma (HCC) from several perspectives. ZBP-89 can upregulate apoptosis in HCC in a p53-dependent or - independent manner. In addition, the negative regulation of ZBP-89 on liver cancer stemness sheds light on its possible effect on sensitizing HCC to chemotherapies and the reduction of HCC relapse. The prognostic significance of ZBP-89 in HCC patients further suggests its clinical importance as a potential tumor suppressor. Expert opinion: Given the roles of ZBP-89 in HCC, we believe, ZBP-89 is a promising therapeutic target for enhancing apoptosis and diminishing the liver cancer stemness. At the same time, we also face a series of challenges, especially in the clinical implication of ZBP-89. Resolving the current controversies will advance the development of ZBP-89 for anti-HCC therapy.

Glioma-associated oncogene homolog 1 (GLI1), an oncogenic molecule in non-small cell lung cancer (NSCLC), promotes the growth of NSCLC by enhancing lung cancer stem cells (LCSCs). However, the mechanism responsible remains unknown. FOXP3 is known to maintain LCSCs. The aim of this study was to explore whether GLI1 enhanced LCSCs via stimulating FOXP3. Experiments were performed in NSCLC tissue samples, cell lines and the animal tumor model. The expression of GLI1- and LCSC-related molecules was assessed at protein and mRNA levels. Relevant cell functions were also determined. A tumor xenograft mouse model was established to confirm the oncogenic role of GLI1. We confirmed that the expression of GLI1 was up-regulated in the tumor tissues of NSCLC compared with adjacent non-tumor tissues. But no significant association between GLI1 and clinicopathological characteristics was found. GLI1 expression was positively correlated with FOXP3 and it could promote FOXP3 expression likely via acting on the promoter of FOXP3. Along with the upregulation of FOXP3, GLI1 increased the expression of LCSC markers, ALDH1A1 and OCT4A, and the formation of tumor spheres, whereas the inhibition of GLI1 decreased the above features. We also found the involvement of Notch1 activation in GLI1-mediated FOXP3 pathway. The In vivo mouse tumor model verified the positive role of GLI1 in the growth of the tumor. Collectively, this study has demonstrated that GLI1 stimulates FOXP3 to promote LCSCs.

Objectives Veterans with laryngeal and oropharyngeal cancer remain an understudied patient population despite a high incidence of disease and decreased survival compared to the general population. Our objective was to evaluate treatment patterns for laryngeal and oropharyngeal cancer in patients treated at one of the Veterans Health Administration's busiest cancer centers in order to generate some basic benchmarks for treatment delivery in the veteran population. Methods We reviewed 338 patients treated at the Michael E. DeBakey Veterans Affairs Medical Center between 2000 and 2012. Results Oropharyngeal site and advanced age were associated with worse overall and disease-free survival. Treatment periods (mean) were as follows: 1) referral-diagnosis, 26 days; 2) diagnosis-surgery, 29 days; and 3) diagnosis-radiation, 58 days. Adjuvant radiation was initiated within 6 weeks of surgery in 42% of patients and 68% of patients had a total treatment package time ≤100 days. Time from diagnosis to treatment initiation, surgery to adjuvant radiation interval and total treatment package time did not impact survival. Conclusions This study establishes basic benchmarks for laryngeal and oropharyngeal cancer treatment delivery in veterans. Additional efforts are warranted to improve consistency and provide treatment in line with NCCN recommendations and literature consensus. Level of Evidence 2b.

Aim: Our study investigated the significance of the crosstalk between long noncoding RNAs (lncRNAs) and mRNAs in gastric cancer (GC). Methods: lncRNA and mRNA expression profiling data in 671 GC tumors and 77 nontumorous gastric tissues were retrieved from the gene expression omnibus database: GSE54129, GSE13911, GSE19826, GSE79973, GSE15459 and GSE66229. Differentially expressed analysis, RNA coexpression network construction, gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) enrichment analyses were conducted in this study. Results: Using differentially expressed and prognostic lncRNAs or mRNAs in GC, we constructed the lncRNA–mRNA coexpression networks. This network involved with vital GO and KEGG pathways. Conclusion: Our study reveals coexpressed lncRNAs and mRNAs as transcriptional hallmarks in GC patients which provide interesting information regarding the incidence and outcome of GC.

Purpose: The spatial heterogeneity of phenotypic and molecular characteristics of CTCs within the circulatory system remains unclear. Herein, we mapped the distribution and characterized biological features of CTCs along the transportation route in hepatocellular carcinoma (HCC). Experimental Design: In 73 localized HCC patients, blood was drawn from peripheral vein (PV), peripheral artery (PA), hepatic veins (HV), infrahepatic inferior vena cava (IHIVC), and portal vein (PoV) before tumor resection. Epithelial and mesenchymal transition (EMT) phenotype in CTCs were analyzed by a 4-channel immunofluorescence CellSearch assay and microfluidic quantitative RT-PCR. The clinical significance of CTCs from different vascular sites was evaluated. Results: The CTC number and size gradient between tumor efferent vessels and postpulmonary peripheral vessels was marked. Tracking the fate of CTC clusters revealed that CTCs displayed an aggregated–singular-aggregated manner of spreading. Single-cell characterization demonstrated that EMT status of CTCs was heterogeneous across different vascular compartments. CTCs were predominantly epithelial at release, but switched to EMT-activated phenotype during hematogeneous transit via Smad2 and β-catenin related signaling pathways. EMT activation in primary tumor correlated with total CTC number at HV, rather than epithelial or EMT-activated subsets of CTCs. Follow-up analysis suggested that CTC and circulating tumor microemboli burden in hepatic veins and peripheral circulation prognosticated postoperative lung metastasis and intrahepatic recurrence, respectively. Conclusions: The current data suggested that a profound spatial heterogeneity in cellular distribution and biological features existed among CTCs during circulation. Multivascular measurement of CTCs could help to reveal novel mechanisms of metastasis and facilitate prediction of postoperative relapse or metastasis pattern in HCC. Clin Cancer Res; 24(3); 547–59. ©2017 AACR.

BACKGROUND: Recurrence or metastasis of hepatocellular carcinoma (HCC) is mainly intrahepatic after curative resection, demonstrating that the peritumoral environment is important but often neglected. Programmed death ligand 1 (PD-L1) in intratumoral liver tissues is a poor prognosis factor whose impact is removed after curative resection. However, PD-L1 expression remains in the peritumoral liver tissues and its distribution and prognostic value are still not clear. METHODS: We assessed the expression of PD-L1 by immunohistochemistry in peritumoral liver tissues from 90 HCC patients who underwent curative hepatectomy. The results were validated in an independent cohort of additional 90 HCC patients. RESULTS: We found PD-L1 positive expression in 31.11% (28/90) of peritumoral tissues. Peritumoral PD-L1 expression was associated with a significantly worse overall survival (OS) (P = .000) and disease-free survival (DFS) (P = .001) compared to the negative expression group. Additionally, peritumoral PD-L1 positivity significantly correlated with vascular invasion and a lower albumin level (≤35 g/L). Univariate and multivariate Cox regression models both revealed peritumoral PD-L1 as an independent prognostic factor for OS (HR = 2.853, P = .002) and DFS (HR = 2.362, P = .003). The prognostic value of PD-L1 positivity was validated in the independent data set. CONCLUSIONS: Our data suggest PD-L1 expression in peritumoral hepatocytes is an independent prognostic factor for OS and DFS. This implies that future anti-cancer therapy should target not only residual tumor cells but also the “soil” for promoting tumor growth. Peritumoral PD-L1 could be a good target for adjuvant therapy after hepatectomy.

Aim: We performed a study to identify the role of microRNA in thymoma. Patients & methods: One hundred twenty-three thymoma patients with clinical information and miRNA expression data from The Cancer Genome Atlas were included in the study. Comprehensive bioinformatics analysis was integrated in our analysis. Results & conclusion: Seven miRNAs were found to be associated with overall survival (p < 0.001). Another four miRNAs were found to be associated with disease-free survival (p < 0.001). Type C thymoma can be distinguished from nontype C thymoma by miRNAs. Interestingly, seven miRNAs showed both prognostic and subtype-specific potential. Our findings suggest that miRNAs can be used for prognostic prediction and subtype stratification.

Recent studies have suggested that segmentectomy may be an acceptable alternative treatment to lobectomy for surgical management of smaller lung adenocarcinomas. The objective of this study was to compare survival after lobectomy and segmentectomy among patients with pathological stage IA adenocarcinoma categorized as stage T1b (0 to ≤20 mm) according to the new eighth edition of the TNM system.In total, 7989 patients were identified from the Surveillance, Epidemiology, and End Results registry. Propensity scores generated from logistic regression on preoperative characteristics were used to balance the selection bias of undergoing segmentectomy. Overall and lung cancer-specific survival rates of patients undergoing segmentectomy and lobectomy were compared in propensity score-matched groups.Overall, 564 patients (7.1%) underwent segmentectomy. Lobectomy led to better overall and lung cancer-specific survival than segmentectomy for the entire cohort (log-rank p0.01). After 1:2 propensity score matching, segmentectomy (n = 552) was no longer associated with significantly worse overall survival (5-year survival = 74.45% versus 76.67%, hazard ratio = 1.09, 95% confidence interval: 0.90-1.33) or lung cancer-specific survival (5-year survival = 83.89% versus 86.11%, hazard ratio = 1.12, 95% confidence interval: 0.86-1.46) compared with lobectomy (n = 1085) after adjustment for age, sex, lymph node quantity, and histological subtype. Similar negative findings were identified when patients were stratified according to sex, age, histological subtype, and number of evaluated lymph nodes.Patients who underwent segmentectomy may have survival outcomes no different than those of some patients who received lobectomy for pathological stage IA adenocarcinomas at least 10 but no larger than 20 mm in size. These results should be further confirmed through prospective randomized trials.

Background: Currently, there is a global attempt to identify potential anti-cancer agents with low toxicity. Previous studies have found that glycyrrhizin exerts anti-cancer action with low toxicity through suppressing thromboxane A2 (TxA2) in lung cancer cell lines. However, these effects have not yet been determined in animal models of lung cancer. Methods: Human lung adenocarcinoma xenografts were established in nude mice by the introduction of A549 cells with stable transfection of the TxA2 receptor (TPα). The animal model was confirmed by the hematoxylin and eosin (H&E) method. Tumor-bearing mice were then administered graded concentrations of glycyrrhizin, cisplatin or both. After the treatments, body weights of all animals were recorded, and immunohistochemistry staining of lung tissues and serum biochemistry detection of aspartate amino transferase (AST), alanine amino transferase (ALT), urea and creatinine were carried out. Results: Treatment with glycyrrhizin alone or the combination of cisplatin and glycyrrhizin profoundly reduced expression of thromboxane synthase (TxAS) as well as proliferating cell nuclear antigen (PCNA), recovered the body weight, and rescued damage of liver and kidney in tumor-bearing mice. Although it inhibited PCNA expression, cisplatin could not significantly suppress TxAS expression. Because of a positive feedback loop between TPα and TxAS, the effects of glycyrrhizin are possibly attributable to the suppression of the TxA2 pathway. Conclusions: This study provides in vivo evidence to support glycyrrhizin as a potential candidate for developing new regimens to overcome tumor progression and the resistance and toxicity of cisplatin.

Lipid oversupply may induce CD36 sarcolemmal translocation to facilitate fatty acid transport, which in turn causes dyslipidemia and type 2 diabetes. However, the underlying mechanisms of CD36 redistribution are still yet to be unraveled. Methods: High fat diet fed mice and palmitate/oleic acid-treated L6 cells were used to investigate the initial events of subcellular CD36 recycling prior to insulin resistance. The regulation of CD36 sarcolemmal translocation by lipid oversupply was assessed by insulin tolerance test (ITT), oral glucose tolerance test (OGTT), glucose/fatty acid uptake assay, surface CD36 and GLUT4 detection, and ELISA assays. To elucidate the underlying mechanisms, specific gene knockout, gene overexpression and/or gene inhibition were employed, followed by Western blot, co-immunoprecipitation, immunostaining, and kinase activity assay. Results: Upon lipid/fatty acid overload, PKCζ activity and TBC1D1 phosphorylation were enhanced along with increased sarcolemmal CD36. The inhibition of PKCζ or TBC1D1 was shown to block fatty acid-induced CD36 translocation and was synergistic in impairing CD36 redistribution. Mechanically, we revealed that AMPK was located upstream of PKCζ to control its activity whereas Rac1 facilitated PKCζ translocation to the dorsal surface of the cell to cause actin remodeling. Furthermore, AMPK phosphorylated TBC1D1 to release retained cytosolic CD36. The activated PKCζ and phosphorylated TBC1D1 resulted in a positive feedback regulation of CD36 sarcolemmal translocation. Conclusion: Collectively, our study demonstrated exclusively that lipid oversupply induced CD36 sarcolemmal translocation via dual modulation of PKCζ and TBC1D1, which was as an early event prior to insulin resistance. The acquired data may provide potential therapy targets to prevent lipid oversupply-induced insulin resistance.

Background Epidemiological observations have demonstrated that ambient fine particulate matter with dp

Background: Early data showed that FOXP3 could induce epithelial-mesenchymal transition by stimulating the Wnt/β-catenin signaling pathway in non-small cell lung cancer (NSCLC). However, how the expression of FOXP3 is regulated in NSCLC remains unknown. We thus explored the impacts of the long noncoding RNA EGFR antisense RNA 1 (EGFR-AS1) and hypoxia-inducible factor-2A (HIF2A) on FOXP3 expression and the cancer stemness of NSCLC. Methods: Lung tissues samples from 87 patients with NSCLC and two NSCLC cell lines were used in this study. The regulation of FOXP3 and lung cancer cell stemness by EGFR-AS1 and HIF2A was determined at molecular levels in NSCLC tissue samples and cultured cells in the presence/absence of the smoking carcinogen, 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK) (also known as nicotine-derived nitrosamine ketone). The results were confirmed in tumor xenograft models. Results: We found that NNK decreased the expression of EGFR-AS1 in the long term, but increased the expression of HIF2A and FOXP3 to stimulate lung cancer cell stemness. EGFR-AS1 significantly inhibited FOXP3 expression and NSCLC cell stemness, whereas HIF2A obviously promoted both. The enhancement of lung cancer stemness by FOXP3 was, at least partially, via stimulating Notch1, as the inhibition of Notch1 could markedly diminish the effect of FOXP3. Conclusions: FOXP3, the expression of which is under the fine control of EGFR-AS1, is a critical molecule that promotes NSCLC cancer cell stemness through stimulating the Notch1 pathway.

The poor clinical outcome of hepatocellular carcinoma (HCC) patients is ascribed to the resistance of HCC cells to traditional treatments and tumor recurrence after curative therapies. Cancer stem cells (CSCs) have been identified as a small subset of cancer cells which have high capacity for self-renewal, differentiation and tumorigenesis. Recent advances in the field of liver CSCs (LCSCs) have enabled the identification of CSC surface markers and the isolation of CSC subpopulations from HCC cells. Given their central role in cancer initiation, metastasis, recurrence and therapeutic resistance, LCSCs constitute a therapeutic opportunity to achieve cure and prevent relapse of HCC. Thus, it is necessary to develop therapeutic strategies to selectively and efficiently target LCSCs. Small molecular inhibitors targeting the core stemness signaling pathways have been actively pursued and evaluated in preclinical and clinical studies. Other alternative therapeutic strategies include targeting LCSC surface markers, interrupting the CSC microenvironment, and altering the epigenetic state. In this review, we summarize the properties of CSCs in HCC and discuss novel therapeutic strategies that can be used to target LCSCs.