Thyroid hormone negatively regulates tumorigenesis through suppression of BC200.

Thyroid hormone (T3) and its receptor (TR) are involved in cancer progression. While deregulation of long non-coding RNA (lncRNA) expression has been detected in many tumor types, the mechanisms underlying specific involvement of lncRNAs in tumorigenicity remain unclear. Experiments from the current study revealed negative regulation of BC200 expression by T3/TR. BC200 was highly expressed in hepatocellular carcinoma (HCC) and effective as an independent prognostic marker. BC200 promoted cell growth and tumor sphere formation, which was mediated via regulation of cell cycle-related genes and stemness markers. Moreover, BC200 protected cyclin E2 mRNA from degradation. Cell growth ability was repressed by T3, but partially enhanced upon BC200 overexpression. Mechanistically, BC200 directly interacted with cyclin E2 and promoted CDK2-cyclin E2 complex formation. Upregulation of cell cycle-related genes in hepatoma samples was positively correlated with BC200 expression. Our collective findings support the utility of a potential therapeutic strategy involving targeting of BC200 for the treatment of HCC.

The Long Non-Coding RNA MIR503HG Enhances Proliferation of Human ALK-Negative Anaplastic Large-Cell Lymphoma.

Anaplastic lymphoma kinase (ALK)-negative anaplastic large-cell lymphoma (ALCL) is a rare type of highly malignant, non-Hodgkin lymphoma. Currently, only a few gene rearrangements have been linked to ALK-negative ALCL progression. However, the specific molecular mechanisms underlying the growth of ALK-negative ALCL tumors remain unclear. Here, we investigated aberrantly expressed, long non-coding RNAs (lncRNAs) in ALK-negative ALCL and assessed their potential biological function. MIR503HG (miR-503 host gene) was highly expressed in ALK-negative cell lines and was significantly upregulated in tumors in mice formed from ALK-negative ALCL cell lines. Depletion of MIR503HG suppressed tumor cell proliferation in vivo and in vitro; conversely, its overexpression enhanced tumor cell growth. MIR503HG-induced proliferation was mediated by the induction of microRNA-503 (miR-503) and suppression of Smurf2, resulting in stabilization of the tumor growth factor-ß receptor (TGFBR) and enhanced tumor cell growth. Collectively, these findings support a potential role for MIR503HG in cancer cell proliferation through the miR-503/Smurf2/TGFBR axis and indicate that MIR503HG is a potential marker in ALK-negative ALCL.

Stimulation of Interferon-Stimulated Gene 20 by Thyroid Hormone Enhances Angiogenesis in Liver Cancer.

Thyroid hormone, 3,3',5-triiodo-l-thyronine (T3), mediates several physiological processes, including embryonic development, cellular differentiation and cell proliferation, via binding to its nuclear thyroid receptors (TR). Previous microarray and Chromatin immunoprecipitation (ChIP)-on-ChIP analyses have revealed that interferon-stimulated gene 20 kDa (ISG20), an exoribonuclease involved in the antiviral function of interferon, is up-regulated by T3 in HepG2-TR cells. However, the underlying mechanisms of ISG20 action in tumor progression remain unknown to date. Here, we verified induction of ISG20 mRNA and protein expression by T3 in HepG2-TR cells. Based on the ChIP-on-ChIP database, potential thyroid hormone responsive element of the ISG20 promoter region was predicted, and the result confirmed with the ChIP assay. Functional assays showed that forced expression of ISG20 leads to significant promotion of metastasis and angiogenesis, both in vitro and in vivo. Furthermore, the angiogenic-related protein, interleukin-8 (IL-8), was up-regulated through a T3-mediated increase in ISG20, as determined using a human angiogenesis array kit. Induction of IL-8 signaling activated the p-JAK2/p-STAT3 pathway, in turn, leading to promotion of tumor metastasis and angiogenesis. Furthermore, ISG20 overexpression in hepatocellular carcinoma (HCC) specimens was positively correlated with clinical parameters, including vascular invasion, α-fetoprotein and tumor size. Higher ISG20 expression was significantly correlated with poorer recurrence-free survival in HCC patients. Our results collectively indicate higher TR-dependent expression of ISG20 in a subset of HCC, supporting an oncogenic role in HCC progression.

Taurine up-regulated gene 1 functions as a master regulator to coordinate glycolysis and metastasis in hepatocellular carcinoma.

Cancer cells display altered glucose metabolism characterized by a preference for aerobic glycolysis. The aerobic glycolytic phenotype of hepatocellular carcinoma (HCC) is often correlated with tumor progression and poorer clinical outcomes. However, the issue of whether glycolytic metabolism influences metastasis in HCC remains unclear. In the current study, we showed that knockdown of taurine up-regulated gene 1 (TUG1) induces marked inhibition of cell migration, invasion, and glycolysis through suppression of microRNA (miR)-455-3p. MiR-455-3p, which is transcriptionally repressed by p21, directly targets the 3' untranslated region of adenosine monophosphate-activated protein kinase subunit beta 2 (AMPKß2). The TUG1/miR-455-3p/AMPKß2 axis regulates cell growth, metastasis, and glycolysis through regulation of hexokinase 2 (HK2). TUG1 is clearly associated with HK2 overexpression and unfavorable prognosis in HCC patients. CONCLUSION: Our data collectively highlight that novel regulatory associations among TUG1, miR-455-3p, AMPKß2, and HK2 are an important determinant of glycolytic metabolism and metastasis in HCC cells and support the potential utility of targeting TUG1/HK2 as a therapeutic strategy for HCC. (Hepatology 2018;67:188-203).

The long non-coding RNA LOC441204 enhances cell growth in human glioma.

Glioma is the most common and aggressive type of brain tumor. While long non-coding RNAs (lncRNAs) are clearly more abundant in human brain than protein-coding genes, the specific roles of lncRNAs and mechanisms underlying their dysregulation in glioma remain unclear. Here, we focused on lncRNAs that are differentially expressed in brain tumor and their potential biological functions. LOC441204, a novel non-coding RNA gene displaying high expression in clinical specimens of brain tumor and significant upregulation in glioma cell lines in microarray analyses, was selected for further study. Notably, knockdown of LOC441204 suppressed tumor cell proliferation in two glioma cell lines. Moreover, LOC441204-induced tumor cell growth was mediated the stabilization of ß-catenin pathway. Briefly, LOC441204 bound to ß-catenin preventing its degradation, resulting in downstream p21 repression and cdk4 activation to enhance glioma cell proliferation. Collectively, our findings indicate a pro-oncogenic role of LOC441204 in tumor cell growth through activation of the ß-catenin/p21/cdk4 cascade to act as a potential diagnostic marker or therapeutic target in brain tumor.

The long non-coding RNA LINC01013 enhances invasion of human anaplastic large-cell lymphoma.

Anaplastic large-cell lymphoma (ALCL) is a rare type of highly malignant, non-Hodgkin lymphoma (NHL). Currently, only studies on the chimeric oncogene NPM-ALK have reported a link to ALCL progression. However, the specific molecular mechanisms underlying the invasion of ALCL are still unclear. Here, we sought to investigate differentially expressed, long non-coding RNAs (lncRNAs) in ALCL and their potential biological function. Our microarray analyses revealed that LINC01013, a novel non-coding RNA gene, was highly expressed in clinical specimens of ALCL and was significantly upregulated in invasive ALCL cell lines. Knockdown of LINC01013 suppressed tumor cell invasion; conversely, its overexpression enhanced tumor cell invasion. LINC01013-induced invasion was mediated by activation of the epithelial-to-mesenchymal transition (EMT)-associated proteins, snail and fibronectin. Specifically, LINC01013 induced snail, resulting in activation of fibronectin and enhanced ALCL cell invasion. Collectively, these findings support a potential role for LINC01013 in cancer cell invasion through the snail-fibronectin activation cascade and suggest that LINC01013 could potentially be utilized as a metastasis marker in ALCL.

ChIP-on-chip analysis of thyroid hormone-regulated genes and their physiological significance.

Triiodothyronine (T3) and its receptor (TR) modulate several physiological processes, including cell development, proliferation, differentiation and metabolism. The regulatory mechanism of T3/TR involves binding to the thyroid hormone response element (TRE) within the target gene promoter. However, the number of target genes directly regulated by TRα1 and the specific pathways of TR-regulated target genes remain largely unknown. Here, we expressed TRα1 in a HepG2 cell line and used chromatin immunoprecipitation coupled with microarray to determine the genes that are directly regulated by TRα1 and also involved in cell metabolism and proliferation. Our analysis identified E74-like factor 2 (ELF2), a transcription factor associated with tumor growth, as a direct target downregulated by T3/TR. Overexpression of ELF2 enhanced tumor cell proliferation, and conversely, its knockdown suppressed tumor growth. Additionally, ELF2 restored the proliferative ability of hepatoma cells inhibited by T3/TR. Our findings collectively support a potential role of T3/TR in tumor growth inhibition through regulation of ELF2.

Overexpression of lipocalin 2 in human cervical cancer enhances tumor invasion.

Cervical carcinoma is the third-most common cause of cancer-related deaths in women worldwide. However, the molecular mechanisms underlying the metastasis of cervical cancer are still unclear. Oligonucleotide microarrays coupled with bioinformatics analysis show that cytoskeletal remodeling and epithelial-to- mesenchymal transition (EMT) are significant pathways in clinical specimens of cervical cancer. In accord with clinical observations demonstrating ectopic expression of lipocalin 2 (LCN2), an oncogenic protein associated with EMT, in malignant tumors, was significantly upregulated in cervical cancer and correlated with lymph node metastasis. Overexpression of LCN2 enhanced tumor cell migration and invasion both in vitro and in vivo. Conversely, knockdown or neutralization of LCN2 reduced tumor cell migration and invasion. LCN2-induced migration was stimulated by activation of the EMT-associated proteins, Snail, Twist, N-cadherin, fibronectin, and MMP-9. Our findings collectively support a potential role of LCN2 in cancer cell invasion through the EMT pathway and suggest that LCN2 could be effectively utilized as a lymph node metastasis marker in cervical cancer.

Thyroid hormone-mediated regulation of lipocalin 2 through the Met/FAK pathway in liver cancer.

The thyroid hormone, 3,3',5-triiodo-L-thyronine (T3), regulates cell growth, development and differentiation via interactions with thyroid hormone receptors (TR), but the mechanisms underlying T3-mediated modulation of cancer progression are currently unclear. Lipocalin 2 (LCN2), a tumor-associated protein, is overexpressed in a variety of cancer types. Oligonucleotide microarray, coupled with proteomic analysis, has revealed that LCN2 is positively regulated by T3/TR. However, the physiological role and pathway of T3-mediated regulation of LCN2 in hepatocellular carcinogenesis remain to be characterized. Upregulation of LCN2 after T3 stimulation was observed in a time- and dose-dependent manner. Additionally, TRE on the LCN2 promoter was identified at positions -1444/-1427. Overexpression of LCN2 enhanced tumor cell migration and invasion, and conversely, its knockdown suppressed migration and invasion, both in vitro and in vivo. LCN2-induced migration occurred through activation of the Met/FAK cascade. LCN2 was overexpressed in clinical hepatocellular carcinoma (HCC) patients, compared with normal subjects, and positively correlated with TRα levels. Both TRα and LCN2 showed similar expression patterns in relation to survival rate, tumor grade, tumor stage and vascular invasion. Our findings collectively support a potential role of T3/TR in cancer progression through regulation of LCN2 via the Met/FAK cascade. LCN2 may thus be effectively utilized as a novel marker and therapeutic target in HCC.

A novel small-form NEDD4 regulates cell invasiveness and apoptosis to promote tumor metastasis.

Despite numerous investigations on metastasis, the determinants of metastatic processes remain unclear. We aimed to identify the metastasis-associated genes in hepatocellular carcinoma (HCC). Potent metastatic SK-hep-1 (SK) cells, designated 'SKM', were generated using Transwell assay followed by selection in a mouse model. Genes expressed differentially in SKM and SK cells were identified via microarray analyses. A small form of Neural precursor cell-expressed developmentally downregulated 4 (sNEDD4) was identified to be overexpressed in SKM cells, which was confirmed as a novel transcript using liquid chromatography-mass spectrometry. In clinical specimens, sNEDD4 was significantly overexpressed in tumors and serves as a poor prognostic factor for male patients with HCC (P = 0.035). Upon subcutaneous introduction of sNEDD4-overexpressing SK cells into flanks of nude mice, tumors grew faster than those of the control group. Furthermore, sNEDD4-mediated promotion of tumor metastasis was demonstrated in the orthotopic mouse model. Overexpression of sNEDD4 increased the invasive ability of SK cells through upregulation of matrix metalloproteinase 9 and inhibited serum deprivation-induced apoptosis via upregulation of myeloid cell leukemia 1. In conclusion, sNEDD4 is a novel metastasis-associated gene, which prevents apoptosis under nutrient restriction conditions. The present findings clearly support the prognostic potential of sNEDD4 for HCC.

Repression of microRNA-130b by thyroid hormone enhances cell motility.

BACKGROUND & AIMS: Thyroid hormone (T3) and its receptor (TR) are involved in cell growth and cancer progression. Although deregulation of microRNA (miRNA) expression has been detected in many tumor types, the mechanisms underlying functional impairment and specific involvement of miRNAs in tumor metastasis remain unclear. In the current study, we aimed to elucidate the involvement of deregulated miRNA-130b (miR-130b) and its target genes mediated by T3/TR in cancer progression. METHODS: Quantitative reverse transcription-PCR, luciferase and chromatin immunoprecipitation assays were performed to identify the miR-130b transcript and the mechanisms implicated in its regulation. The effects of miR-130b on hepatocellular carcinoma (HCC) invasion were further examined in vitro and in vivo. Clinical correlations among miR-130b, TRs and interferon regulatory factor 1 (IRF1) were examined in HCC samples using Spearman correlation analysis. RESULTS: Our experiments disclosed negative regulation of miR-130b expression by T3/TR. Overexpression of miR-130b led to marked inhibition of cell migration and invasion, which was mediated via suppression of IRF1. Cell migration ability was promoted by T3, but partially suppressed upon miR-130b overexpression. Furthermore, miR-130b suppressed expression of epithelial-mesenchymal transition (EMT)-related genes, matrix metalloproteinase-9, phosphorylated mammalian target of rapamycin (mTOR), p-ERK1/2, p-AKT and p-signal transducer and activator of transcription (STAT)-3. Notably, miR-130b was downregulated in hepatoma samples and its expression patterns were inversely correlated with those of TRα1 and IRF1. CONCLUSIONS: Our data collectively highlight a novel pathway interlinking T3/TR, miR-130b, IRF1, the EMT-related genes, p-mTOR, p-STAT3 and the p-AKT cascade, which regulates the motility and invasion of hepatoma cells.

Thyroid hormone enhanced human hepatoma cell motility involves brain-specific serine protease 4 activation via ERK signaling.

BACKGROUND: The thyroid hormone, 3, 3', 5-triiodo-L-thyronine (T3), has been shown to modulate cellular processes via interactions with thyroid hormone receptors (TRs), but the secretory proteins that are regulated to exert these effects remain to be characterized. Brain-specific serine protease 4 (BSSP4), a member of the human serine protease family, participates in extracellular matrix remodeling. However, the physiological role and underlying mechanism of T3-mediated regulation of BSSP4 in hepatocellular carcinogenesis are yet to be established. METHODS: The thyroid hormone response element was identified by reporter and chromatin immunoprecipitation assays. The cell motility was analyzed via transwell and SCID mice. The BSSP4 expression in clinical specimens was examined by Western blot and quantitative reverse transcription polymerase chain reaction. RESULTS: Upregulation of BSSP4 at mRNA and protein levels after T3 stimulation is a time- and dose-dependent manner in hepatoma cell lines. Additionally, the regulatory region of the BSSP4 promoter stimulated by T3 was identified at positions -609/-594. BSSP4 overexpression enhanced tumor cell migration and invasion, both in vitro and in vivo. Subsequently, BSSP4-induced migration occurs through the ERK 1/2-C/EBPß-VEGF cascade, similar to that observed in HepG2-TRα1 and J7-TRα1 cells. BSSP4 was overexpressed in clinical hepatocellular carcinoma (HCC) patients, compared with normal subjects, and positively associated with TRα1 and VEGF to a significant extent. Importantly, a mild association between BSSP4 expression and distant metastasis was observed. CONCLUSIONS: Our findings collectively support a potential role of T3 in cancer cell progression through regulation of the BSSP4 protease via the ERK 1/2-C/EBPß-VEGF cascade. BSSP4 may thus be effectively utilized as a novel marker and anti-cancer therapeutic target in HCC.

MicroRNA-196a/-196b promote cell metastasis via negative regulation of radixin in human gastric cancer.

MicroRNAs (miRNAs) play an important role to contribute carcinogenesis. The aim of the current study was to identify useful biomarkers from miRNAs. Differential miRNA profiles were analyzed using the miRNA qRT-PCR-based assay. Two of the most upregulated miRNAs were selected and validated. The miR-196a/-196b levels were significantly increased in gastric cancer (GC) tissues (n=109). Overexpression of miR-196a/-196b was significantly associated with tumor progression and poorer 5-year survival outcomes. Overexpression of miR-196a/-196b enhances GC cell migration and invasion. Further, radixin was identified as a target gene of miR-196a/-196b. Elevated miR-196a/-196b expression in GC cells led to reduced radixin protein levels and vice versa. Notably, an inverse correlation between miR-196a/-196b and radixin mRNA and protein expression was observed in GC tissues with in situ hybridization and immunohistochemistry analyses. Together, miR-196a/-196b inhibitory oligonucleotides or overexpression of the radixin may thus have therapeutic potential in suppressing GC metastasis.

Thyroid hormone receptor inhibits hepatoma cell migration through transcriptional activation of Dickkopf 4.

Triiodothyronine (T3) is a potent form of thyroid hormone mediates several physiological processes including cellular growth, development, and differentiation via binding to the nuclear thyroid hormone receptor (TR). Recent studies have demonstrated critical roles of T3/TR in tumor progression. Moreover, long-term hypothyroidism appears to be associated with the incidence of human hepatocellular carcinoma (HCC), independent of other major HCC risk factors. Dickkopf (DKK) 4, a secreted protein that antagonizes the canonical Wnt signaling pathway, is induced by T3 at both mRNA and protein levels in HCC cell lines. However, the mechanism underlying T3-mediated regulation of DKK4 remains unknown. In the present study, the 5' promoter region of DKK4 was serially deleted, and the reporter assay performed to localize the T3 response element (TRE). Consequently, we identified an atypical direct repeat TRE between nucleotides -1645 and -1629 conferring T3 responsiveness to the DKK4 gene. This region was further validated using chromatin immunoprecipitation (ChIP) and electrophoretic mobility shift assay (EMSA). Stable DKK4 overexpression in SK-Hep-1 cells suppressed cell invasion and metastatic potential, both in vivo andin vitro, via reduction of matrix metalloproteinase-2 (MMP-2) expression. Our findings collectively suggest that DKK4 upregulated by T3/TR antagonizes the Wnt signal pathway to suppress tumor cell progression, thus providing new insights into the molecular mechanism underlying thyroid hormone activity in HCC.

Glyoxalase-I is a novel prognosis factor associated with gastric cancer progression.

Glyoxalase I (GLO1), a methylglyoxal detoxification enzyme, is implicated in the progression of human malignancies. The role of GLO1 in gastric cancer development or progression is currently unclear. The expression of GLO1 was determined in primary gastric cancer specimens using quantitative polymerase chain reaction, immunohistochemistry (IHC), and western blotting analyses. GLO1 expression was higher in gastric cancer tissues, compared with that in adjacent noncancerous tissues. Elevated expression of GLO1 was significantly associated with gastric wall invasion, lymph node metastasis, and pathological stage, suggesting a novel role of GLO1 in gastric cancer development and progression. The 5-year survival rate of the lower GLO1 expression groups was significantly greater than that of the higher expression groups (log rank P = 0.0373) in IHC experiments. Over-expression of GLO1 in gastric cancer cell lines increases cell proliferation, migration and invasiveness. Conversely, down-regulation of GLO1 with shRNA led to a marked reduction in the migration and invasion abilities. Our data strongly suggest that high expression of GLO1 in gastric cancer enhances the metastasis ability of tumor cells in vitro and in vivo, and support its efficacy as a potential marker for the detection and prognosis of gastric cancer.

Overexpression of ADP-ribosylation factor 1 in human gastric carcinoma and its clinicopathological significance.

Gastric cancer is the sixth leading cause of cancer-related death in Taiwan, and the identification of related factors is essential to increase patient survival. ADP-ribosylation factor 1 (ARF1) was initially identified using 2-D electrophoresis combined with MALDI-time-of-flight mass spectrometry. ADP-ribosylation factor 1 belongs to the Ras superfamily or GTP-binding protein family and has been shown to enhance cell proliferation. In the current study, we evaluated the potential of ARF1 as a biomarker for gastric cancer detection. ADP-ribosylation factor 1 mRNA was upregulated in tumor tissues (compared with adjacent non-tumor tissues, n = 55) in approximately 67.2% of gastric cancer patients. Expression of ARF1 protein was additionally observed using Western blot and immunohistochemistry (IHC) analyses. The clinicopathological correlations of ARF1 were further evaluated. Elevated ARF1 expression was strongly correlated with lymph node metastasis (P = 0.008), serosal invasion (P = 0.046), lymphatic invasion (P = 0.035), and pathological staging (P = 0.010). Moreover, the 5-year survival rate for the lower ARF1 expression group (n = 50; IHC score < 90) was higher than that of the higher expression group (n = 60; IHC score ≥ 90) (P = 0.0228, log-rank test). To establish the specific function of ARF1 in human gastric cancer, isogenic ARF1-overexpressing cell lines were prepared. Our results showed that ARF1-overexpressing clones display enhanced cell proliferation, migration, and invasion. Furthermore, ARF1-overexpression might contribute to poor prognosis of patients. These findings collectively support the utility of ARF1 as a novel prognostic marker for gastric cancer and its role in cell invasion.

Dickkopf 4 positively regulated by the thyroid hormone receptor suppresses cell invasion in human hepatoma cells.

UNLABELLED: Thyroid hormone (T(3)) mediates cellular growth, development, and differentiation by binding to the nuclear thyroid hormone receptor (TR). Recent studies suggest that long-term hypothyroidism is associated with human hepatocellular carcinoma (HCC) independent from other major HCC risk factors. Dickkopf (DKK) 4, a secreted protein, antagonizes the Wnt signal pathway. In this study, we demonstrate that T(3) may play a suppressor role by inducing DKK4 expression in HCC cells at both the messenger RNA (mRNA) and protein levels. DKK4 was down-regulated in 67.5% of HCC cancerous tissues. The decrease in DKK4 levels was accompanied by a concomitant decrease in TR protein levels in the matched cancerous tissues in 31% of tissues compared by immunoblotting with the adjacent noncancerous tissues. Further, TR and DKK4 expression levels were positively correlated in both normal and cancerous specimens by tissue array analysis. In function assays, stable DKK4 transfected into J7 or HepG2 cells decreased cell invasion in vitro. Conversely, knocking down DKK4 restores cell invasiveness. DKK4-expressing J7 clones showed increased degradation of ß-catenin, but down-regulation of CD44, cyclin D1, and c-Jun. To investigate the effect of DKK4 and TR on tumor growth in vivo, we established a xenograft of J7 cells in nude mice. J7-DKK4 and J7-TRα1 overexpressing mice, which displayed growth arrest, lower lung colony formation index, and smaller tumor size than in control mice, supporting an inhibitory role of DKK4 in tumor progression. CONCLUSION: Taken together, these data suggest that the TR/DKK4/Wnt/ß-catenin cascade influences the proliferation and migration of hepatoma cells during the metastasis process and support a tumor suppressor role of the TR.