Secreted frizzled-related protein 4 induces gastric cancer progression and resistance to cisplatin and oxaliplatin via ß-catenin dysregulation.

INTRODUCTION: Gastric cancer is the 5th most common cancer and 3rd leading cause of cancer-related death worldwide. There are three main ways to treat gastric cancer: surgical resection, radiation therapy, and drug therapy. Furthermore, combinations of two to three regimens can improve survival. However, the survival outcomes of chemotherapy in advanced gastric cancer patients are still unsatisfactory. Unfortunately, no widely useful biomarkers have been verified to predict the efficacy of chemotherapy for locally advanced gastric cancer. METHODS: An MTT assay was used to determine the cell viability after cisplatin or oxaliplatin treatment. Western blotting and immunohistochemistry were utilized to examine the sFRP4 level and associated signaling pathways. Immunofluorescence staining was utilized to analyze the location of ß-catenin. Colony formation and Transwell assays were used to analyze the functions related with cisplatin, oxaliplatin and sFRP4. RESULTS: We have found that gastric cancer patients treated with combinations of 5-fluorouracil (5-FU) and cisplatin regimens have better survival rates than those treated with 5-FU-based chemotherapy alone. Secreted frizzled-related protein 4 (sFRP4) was selected as a potential target from stringent analysis and intersection of 5-FU and cisplatin resistance-related gene sets. sFRP4 was shown to be overexpressed in clinical gastric tumor tissues and positively correlated with a worse survival rate. In addition, sFRP4 and ß-catenin were upregulated in cisplatin-resistant and oxaliplatin-resistant gastric cancer cells compared to parental cells. Immunofluorescence staining and nuclear fractionation showed that ß-catenin translocated from the cytosol into the nucleus. Moreover, sFRP4 was detected in the conditioned medium of these resistant cells, which indicates that sFRP4 might have an extracellular role in chemotherapy resistance. Increased migration capacity and dysregulation of epithelial-mesenchymal transition-related markers, which might result from the dysregulation of sFRP4, were observed in cisplatin-resistant and oxaliplatin-resistant gastric cancer cells. DISCUSSION/CONCLUSION: In summary, sFRP4 might play a critical role in resistance to cisplatin and oxaliplatin, cell metastasis and poor prognosis in gastric cancer via the Wnt-ß-catenin pathway. Investigations of the molecular mechanism underlying sFRP4-modulated cancer progression and chemotherapeutic outcomes can provide additional therapeutic strategies for gastric cancer.

Functional Role of Mitochondrial DNA in Cancer Progression.

Mitochondrial DNA (mtDNA) has been identified as a significant genetic biomarker in disease, cancer and evolution. Mitochondria function as modulators for regulating cellular metabolism. In the clinic, mtDNA variations (mutations/single nucleotide polymorphisms) and dysregulation of mitochondria-encoded genes are associated with survival outcomes among cancer patients. On the other hand, nuclear-encoded genes have been found to regulate mitochondria-encoded gene expression, in turn regulating mitochondrial homeostasis. These observations suggest that the crosstalk between the nuclear genome and mitochondrial genome is important for cellular function. Therefore, this review summarizes the significant mechanisms and functional roles of mtDNA variations (DNA level) and mtDNA-encoded genes (RNA and protein levels) in cancers and discusses new mechanisms of crosstalk between mtDNA and the nuclear genome.

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).

Argininosuccinate synthetase 1 contributes to gastric cancer invasion and progression by modulating autophagy.

Argininosuccinate synthetase 1 (ASS1) is a rate-limited enzyme in arginine biosynthesis. The oncogenic potential of ASS1 in terms of prognosis and cancer metastasis in arginine prototrophic gastric cancer (GC) remains unclear at present. We identify differentially expressed proteins in microdissected GC tumor cells relative to adjacent nontumor epithelia by isobaric mass tag for relative and absolute quantitation proteomics analysis. GC cells with stable expression or depletion of ASS1 were further analyzed to identify downstream molecules. We investigated their effects on chemoresistance and cell invasion in the presence or absence of arginine. ASS1 was highly expressed in GC and positively correlated with GC aggressiveness and poor outcome. Depletion of ASS1 led to inhibition of tumor growth and decreased cell invasion via induction of autophagy-lysosome machinery, resulting in degradation of active ß-catenin, Snail, and Twist. Ectopic expression of ASS1 in GC cells reversed these effects and protected cancer cells from chemotherapy drug-induced apoptosis via activation of the AKT-mammalian target of rapamycin signaling pathway. ASS1 contributes to GC progression by enhancing aggressive potential resulting from active ß-catenin, Snail, and Twist accumulation. Our results propose that ASS1 might contribute to GC metastasis and support its utility as a prognostic predictor of GC.-Tsai, C.-Y., Chi, H.-C., Chi, L.-M., Yang, H.-Y., Tsai, M.-M., Lee, K.-F., Huang, H.-W., Chou, L.-F., Cheng, A.-J., Yang, C.-W., Wang, C.-S., Lin, K.-H. Argininosuccinate synthetase 1 contributes to gastric cancer invasion and progression by modulating autophagy.

Molecular functions and clinical impact of thyroid hormone-triggered autophagy in liver-related diseases.

The liver is controlled by several metabolic hormones, including thyroid hormone, and characteristically displays high lysosomal activity as well as metabolic stress-triggered autophagy, which is stringently regulated by the levels of hormones and metabolites. Hepatic autophagy provides energy through catabolism of glucose, amino acids and free fatty acids for starved cells, facilitating the generation of new macromolecules and maintenance of the quantity and quality of cellular organelles, such as mitochondria. Dysregulation of autophagy and defective mitochondrial homeostasis contribute to hepatocyte injury and liver-related diseases, such as non-alcoholic fatty liver disease (NAFLD) and liver cancer.Thyroid hormones (TH) mediate several critical physiological processes including organ development, cell differentiation, metabolism and cell growth and maintenance. Accumulating evidence has revealed dysregulation of cellular TH activity as the underlying cause of several liver-related diseases, including alcoholic or non-alcoholic fatty liver disease and liver cancer. Data from epidemiologic, animal and clinical studies collectively support preventive functions of THs in liver-related diseases, highlighting the therapeutic potential of TH analogs. Elucidation of the molecular mechanisms and downstream targets of TH should thus facilitate the development of therapeutic strategies for a number of major public health issues.Here, we have reviewed recent studies focusing on the involvement of THs in hepatic homeostasis through induction of autophagy and their implications in liver-related diseases. Additionally, the potential underlying molecular pathways and therapeutic applications of THs in NAFLD and HCC are discussed.

Impact of DNA and RNA Methylation on Radiobiology and Cancer Progression.

Radiotherapy is a well-established regimen for nearly half the cancer patients worldwide. However, not all cancer patients respond to irradiation treatment, and radioresistance is highly associated with poor prognosis and risk of recurrence. Elucidation of the biological characteristics of radioresistance and development of effective prognostic markers to guide clinical decision making clearly remain an urgent medical requirement. In tumorigenic and radioresistant cancer cell populations, phenotypic switch is observed during the course of irradiation treatment, which is associated with both stable genetic and epigenetic changes. While the importance of epigenetic changes is widely accepted, the irradiation-triggered specific epigenetic alterations at the molecular level are incompletely defined. The present review provides a summary of current studies on the molecular functions of DNA and RNA m6A methylation, the key epigenetic mechanisms involved in regulating the expression of genetic information, in resistance to irradiation and cancer progression. We additionally discuss the effects of DNA methylation and RNA N6-methyladenosine (m6A) of specific genes in cancer progression, recurrence, and radioresistance. As epigenetic alterations could be reversed by drug treatment or inhibition of specific genes, they are also considered potential targets for anticancer therapy and/or radiotherapy sensitizers. The mechanisms of irradiation-induced alterations in DNA and RNA m6A methylation, and ways in which this understanding can be applied clinically, including utilization of methylation patterns as prognostic markers for cancer radiotherapy and their manipulation for anticancer therapy or use as radiotherapy sensitizers, have been further discussed.

Hepatoma cell functions modulated by NEK2 are associated with liver cancer progression.

NEK2 (NIMA-related expressed kinase 2) is a serine/threonine centrosomal kinase that acts as a critical regulator of centrosome structure and function. Aberrant NEK2 activities lead to failure in regulating centrosome duplication. NEK2 overexpression promotes tumorigenesis and is associated with poor prognosis in several cancers. Increased NEK2 expression during the late pathological stage has been detected in the Oncomine liver dataset and hepatocellular carcinoma (HCC) specimens. Elevated NEK2 protein is associated with poor overall survival in patients with HCC. However, the precise roles and mechanisms of NEK2 in liver cancer progression remain largely unknown. An earlier functional study revealed that NEK2 mediates drug resistance (cisplatin or lipo-doxorubicin) via expression of an ABCC10 transporter. Active angiogenesis and metastasis underlie the rapid recurrence and poor survival of HCC. Results from the current study showed that NEK2 mediates tumor growth, metastasis and angiogenesis in vivo. NEK2-mediated drug resistance was blocked by a specific PI3K or AKT inhibitor. Moreover, NEK2 mediated liver cancer cell migration via pAKT/NF-κB signaling and matrix metalloproteinase (MMP) activation. Angiogenesis was induced via the same signaling pathway and IL-8 stimulation. Our findings collectively indicate that NEK2 modulates hepatoma cell functions, including growth, drug resistance, metastasis and angiogenesis via downstream genes activation.

Roles of Long Noncoding RNAs in Recurrence and Metastasis of Radiotherapy-Resistant Cancer Stem Cells.

Radiotherapy is a well-established therapeutic regimen applied to treat at least half of all cancer patients worldwide. Radioresistance of cancers or failure to treat certain tumor types with radiation is associated with enhanced local invasion, metastasis and poor prognosis. Elucidation of the biological characteristics underlying radioresistance is therefore critical to ensure the development of effective strategies to resolve this issue, which remains an urgent medical problem. Cancer stem cells (CSCs) comprise a small population of tumor cells that constitute the origin of most cancer cell types. CSCs are virtually resistant to radiotherapy, and consequently contribute to recurrence and disease progression. Metastasis is an increasing problem in resistance to cancer radiotherapy and closely associated with the morbidity and mortality rates of several cancer types. Accumulating evidence has demonstrated that radiation induces epithelial-mesenchymal transition (EMT) accompanied by increased cancer recurrence, metastasis and CSC generation. CSCs are believed to serve as the basis of metastasis. Previous studies indicate that CSCs contribute to the generation of metastasis, either in a direct or indirect manner. Moreover, the heterogeneity of CSCs may be responsible for organ specificity and considerable complexity of metastases. Long noncoding RNAs (lncRNAs) are a class of noncoding molecules over 200 nucleotides in length involved in the initiation and progression of several cancer types. Recently, lncRNAs have attracted considerable attention as novel critical regulators of cancer progression and metastasis. In the current review, we have discussed lncRNA-mediated regulation of CSCs following radiotherapy, their association with tumor metastasis and significance in radioresistance of cancer.

Potential Diagnostic, Prognostic and Therapeutic Targets of MicroRNAs in Human Gastric Cancer.

Human gastric cancer (GC) is characterized by a high incidence and mortality rate, largely because it is normally not identified until a relatively advanced stage owing to a lack of early diagnostic biomarkers. Gastroscopy with biopsy is the routine method for screening, and gastrectomy is the major therapeutic strategy for GC. However, in more than 30% of GC surgical patients, cancer has progressed too far for effective medical resection. Thus, useful biomarkers for early screening or detection of GC are essential for improving patients' survival rate. MicroRNAs (miRNAs) play an important role in tumorigenesis. They contribute to gastric carcinogenesis by altering the expression of oncogenes and tumor suppressors. Because of their stability in tissues, serum/plasma and other body fluids, miRNAs have been suggested as novel tumor biomarkers with suitable clinical potential. Recently, aberrantly expressed miRNAs have been identified and tested for clinical application in the management of GC. Aberrant miRNA expression profiles determined with miRNA microarrays, quantitative reverse transcription-polymerase chain reaction and next-generation sequencing approaches could be used to establish sample specificity and to identify tumor type. Here, we provide an up-to-date summary of tissue-based GC-associated miRNAs, describing their involvement and that of their downstream targets in tumorigenic and biological processes. We examine correlations among significant clinical parameters and prognostic indicators, and discuss recurrence monitoring and therapeutic options in GC. We also review plasma/serum-based, GC-associated, circulating miRNAs and their clinical applications, focusing especially on early diagnosis. By providing insights into the mechanisms of miRNA-related tumor progression, this review will hopefully aid in the identification of novel potential therapeutic targets.

Negative modulation of the epigenetic regulator, UHRF1, by thyroid hormone receptors suppresses liver cancer cell growth.

The thyroid hormone, 3,3',5-triiodo-l-thyronine (T3 ), mediates several physiological processes, including embryonic development, cellular differentiation, metabolism and regulation of cell proliferation. Thyroid hormone (T3 ) and its receptor (TR) are involved in metabolism and growth. In addition to their developmental and metabolic functions, TRs play a tumor suppressor role, and therefore, their aberrant expression can lead to tumor transformation. Aberrant epigenetic silencing of tumor suppressor genes promotes cancer progression. The epigenetic regulator, Ubiquitin-like with PHD and ring finger domains 1 (UHRF1), is overexpressed in various cancers. In our study, we demonstrated that T3 negatively regulates UHRF1 expression, both in vitro and in vivo. Our results further indicate that UHRF1 regulation by T3 is indirect and mediated by Sp1. Sp1-binding elements of UHRF1 were identified at positions -664/-505 of the promoter region using the luciferase and chromatin immunoprecipitation assays. Notably, UHRF1 and Sp1 levels were elevated in subgroups of hepatocellular carcinoma patients and inversely correlated with TRα1 expression. Knockdown of UHRF1 expression should therefore provide a means to inhibit hepatoma cell proliferation. Expression of UHRF1 was downregulated by TRs, in turn, relieving silencing of the UHRF1 target gene, p21. Based on the collective findings, we propose that T3 /TR signaling induces hepatoma cell growth inhibition via UHRF1 repression.

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.

Biological significance of a thyroid hormone-regulated secretome.

The thyroid hormone, 3,3,5-triiodo-L-thyronine (T3), modulates several physiological processes, including cellular growth, differentiation, metabolism and proliferation, via interactions with thyroid hormone response elements (TREs) in the regulatory regions of target genes. Several intracellular and extracellular protein candidates are regulated by T3. Moreover, T3-regulated secreted proteins participate in physiological processes or cellular transformation. T3 has been employed as a marker in several disorders, such as cardiovascular disorder in chronic kidney disease, as well as diseases of the liver, immune system, endocrine hormone metabolism and coronary artery. Our group subsequently showed that T3 regulates several tumor-related secretory proteins, leading to cancer progression via alterations in extracellular matrix proteases and tumor-associated signaling pathways in hepatocellular carcinomas. Therefore, elucidation of T3/thyroid hormone receptor-regulated secretory proteins and their underlying mechanisms in cancers should facilitate the identification of novel therapeutic targets. This review provides a detailed summary on the known secretory proteins regulated by T3 and their physiological significance. This article is part of a Special Issue entitled: An Updated Secretome.

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.

Stable isotope labeling with amino acids in cell culture (SILAC)-based quantitative proteomics study of a thyroid hormone-regulated secretome in human hepatoma cells.

The thyroid hormone, 3, 3',5-triiodo-l-thyronine (T(3)), regulates cell growth, development, differentiation, and metabolism via interactions with thyroid hormone receptors (TRs). However, the secreted proteins that are regulated by T(3) are yet to be characterized. In this study, we used the quantitative proteomic approach of stable isotope labeling with amino acids in cell culture coupled with nano-liquid chromatography-tandem MS performed on a LTQ-Orbitrap instrument to identify and characterize the T(3)-regulated proteins secreted in human hepatocellular carcinoma cell lines overexpressing TRα1 (HepG2-TRα1). In total, 1742 and 1714 proteins were identified and quantified, respectively, in three independent experiments. Among these, 61 up-regulated twofold and 11 down-regulated twofold proteins were identified. Eight proteins displaying increased expression and one with decreased expression in conditioned media were validated using Western blotting. Real-time quantitative RT-PCR further disclosed induction of plasminogen activator inhibitor-1 (PAI-1), a T(3) target, in a time-course and dose-dependent manner. Serial deletions of the PAI-1 promoter region and subsequent chromatin immunoprecipitation assays revealed that the thyroid hormone response element on the promoter is localized at positions -327/-312. PAI-1 overexpression enhanced tumor growth and migration in a manner similar to what was seen when T(3) induced PAI-1 expression in J7-TRα1 cells, both in vitro and in vivo. An in vitro neutralizing assay further supported a crucial role of secreted PAI-1 in T(3)/TR-regulated cell migration. To our knowledge, these results demonstrate for the first time that proteins involved in the urokinase plasminogen activator system, including PAI-1, uPAR, and BSSP4, are augmented in the extra- and intracellular space of T(3)-treated HepG2-TRα1 cells. The T(3)-regulated secretome generated in the current study may provide an opportunity to establish the mechanisms underlying T(3)-associated tumor progression and prognosis.

RNASE4 promotes malignant progression and chemoresistance in hypoxic glioblastoma via activation of AXL/AKT and NF-κB/cIAPs signaling pathways.

Glioblastoma (GBM) is the most malignant brain tumor frequently characterized by a hypoxic microenvironment. In this investigation, we unveiled unprecedented role of Ribonuclease 4 (RNASE4) in GBM pathogenesis through integrative methodologies. Leveraging The Cancer Genome Atlas (TCGA) dataset and clinical specimens from normal brain tissues, low- and high-grade gliomas, alongside rigorous in vitro and in vivo functional analyses, we identified a consistent upregulation of RNASE4 correlating with advanced GBM pathological stages and poor clinical survival outcomes. Functional assays corroborated the pivotal influences of RNASE4 on key tumorigenic processes such as cell proliferation, migration, invasion, stemness properties and temozolomide (TMZ) resistance. Further, Gene Set Enrichment Analysis (GSEA) illuminated the involvement of RNASE4 in modulating epithelial-mesenchymal transition (EMT) via activation of AXL, AKT and NF-κB signaling pathways. Furthermore, recombinant human RNASE4 (hRNASE4)-mediated NF-κB activation through IκBα phosphorylation and degradation could result in the upregulation of inhibitors of apoptosis proteins (IAPs), such as cIAP1, cIAP2, and SURVIVIN. Notably, treating RNASE4-induced TMZ-resistant cells with the SURVIVIN inhibitor YM-155 significantly restored cellular sensitivity to TMZ therapy. Herein, this study positions RNASE4 as a potent prognostic biomarker and therapeutic target, offering new insights into molecular pathogenesis of GBM and new avenues for future therapeutic interventions.

CCL16 is a pro-tumor chemokine that recruits monocytes and macrophages to promote hepatocellular carcinoma progression.

Intricate signaling cascades involving chemokines and their cognate receptors on neoplastic and immune constituents within tumor microenvironment have garnered substantial research interest. Our investigation delineates the contribution of Chemokine (C-C motif) ligand 16 (CCL16) to the clinico-pathological features and tumorigenesis of hepatocellular carcinoma (HCC). Analysis of 237 pairs of HCC specimens unraveled a significant association between CCL16 expression and vascular invasion, early-stage clinicopathological features, and diminished recurrence-free survival among HCC patients. Immunohistochemical (IHC) assays of the clinical HCC specimens indicated elevated CCL16 in tumorous versus normal hepatic tissues. Our in vivo experiments demonstrated CCL16 overexpression fostered tumor proliferation, whereas in vitro assays elucidated that CCL16-mediated chemotactic recruitment of monocytes and M2 macrophages was orchestrated via CCR1 and CCR5. In contrast to previous claims that CCL16 is physiologically irrelevant and has minimal affinity for its receptors (CCR1, CCR2, CCR5, CCR8), our findings unravel that inhibition of CCL16/CCR1 and CCL16/CCR5 interactions through receptor-specific antagonists markedly impeded CCL16-directed chemotaxis, migration, adhesion, and leukocyte recruitment. Moreover, CCL16-overexpression in HCCs significantly augmented levels of several cytokines implicated in tumor progression, namely IL-6, IL-10 and VEGFA. IHC analysis of CCL16-overexpressing xenografts elicited greatly enhanced levels of VEGFA and IL-6, while assessments of HCC specimens confirmed a positive correlation between CCL16 expression and IL-6 and VEGFA levels. Collectively, our study highlights oncogenic role of CCL16 in hepatocarcinogenesis and provides a foundational basis for novel therapeutic interventions targeting the CCL16/CCR1/CCR5 axis.

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.

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.

YTHDF3 Modulates EGFR/ATK/ERK/p21 Signaling Axis to Promote Cancer Progression and Osimertinib Resistance of Glioblastoma Cells.

BACKGROUND/AIM: Despite recent advances in EGFR-tyrosine kinase inhibitor (TKI) drugs for glioblastoma multiforme (GBM), intrinsic EGFR alterations in GBM have resulted in drug resistance and unsatisfactory clinical development of EGFR-TKIs. Determining the unknown mechanisms underlying EGFR-TKI drug resistance is an urgent, but unmet, medical need for GBM. Although several m6A RNA methylation regulators, such as reader YTHDF1/2, were recently predicted to be related to GBM recurrence, none was associated with resistance to the 3rd generation EGFR-TKI osimertinib. MATERIALS AND METHODS: Osimertinib-resistant GBM cells (U87OSR) were established to ascertain the correlation between m6A expression and osimertinib resistance, prior to systemic analyses on m6A writers, erasers, and readers. YTHDF3-silencing was employed to reveal changes in IC50, cellular migration, cancer stemness, and p21-guided senescence in U87OSR cells. Signaling pathways and an in vivo xenograft model of U87OSR cells were investigated to delineate the influence of osimertinib-resistance and elevated YTHDF3 expression. RESULTS: YTHDF3 played a crucial role in inducing cellular proliferation, migration, and stemness in U87OSR GBM cells. Importantly, silencing of YTHDF3 markedly reduced the activation of certain signaling pathways, including EGFR- or ITGA7- AKT, and ERK in U87OSR cells. Our study also revealed the oncogenic function of YTHDF3 in inducing senescence escape via p21 down-regulation. In contrast, silencing of YTHDF3 resulted in increased p21 expression, senescence, and suppressed tumor growth in our osimertinib-resistant preclinical model. CONCLUSION: Overall, our research underscores the novel potential of YTHDF3 as a new pharmacological target in GBM treatment, specifically for patients with osimertinib-resistant or refractory tumors.

Cisplatin or Doxorubicin Reduces Cell Viability via the PTPIVA3-JAK2-STAT3 Cascade in Hepatocellular Carcinoma.

Introduction: Hepatocellular carcinoma (HCC) accounts for 80% of all liver cancers and is the 2nd leading cause of cancer-related death in Taiwan. Various factors, including rapid cell growth, a high recurrence rate and drug resistance, make HCC difficult to cure. Moreover, the survival rate of advanced HCC patients treated with systemic chemotherapy remains unsatisfactory. Hence, the identification of novel molecular targets and the underlying mechanisms of chemoresistance in HCC and the development more effective therapeutic regimens are desperately needed. Methods: An MTT assay was used to determine the cell viability after cisplatin or doxorubicin treatment. Western blotting, qRT‒PCR and immunohistochemistry were utilized to examine the protein tyrosine phosphatase IVA3 (PTP4A3) level and associated signaling pathways. ELISA was utilized to analyze the levels of the inflammatory cytokine IL-6 influenced by cisplatin, doxorubicin and PTP4A3 silencing. Results: In this study, we found that PTP4A3 in the cisplatin/doxorubicin-resistant microarray was closely associated with the overall and recurrence-free survival rates of HCC patients. Cisplatin or doxorubicin significantly reduced cell viability and decreased PTP4A3 expression in hepatoma cells. IL-6 secretion increased with cisplatin or doxorubicin treatment and after PTP4A3 silencing. Furthermore, PTP4A3 was highly expressed in tumor tissues versus adjacent normal tissues from HCC patients. In addition, we evaluated the IL-6-associated signaling pathway involving STAT3 and JAK2, and the levels of p-STAT3, p-JAK2, STAT3 and JAK2 were obviously reduced with cisplatin or doxorubicin treatment in HCC cells using Western blotting and were also decreased after silencing PTP4A3. Collectively, we suggest that cisplatin or doxorubicin decreases HCC cell viability via downregulation of PTP4A3 expression through the IL-6R-JAK2-STAT3 cascade. Discussion: Therefore, emerging evidence provides a deep understanding of the roles of PTP4A3 in HCC cisplatin/doxorubicin chemoresistance, which can be applied to develop early diagnosis strategies and reveal prognostic factors to establish novel targeted therapeutics to specifically treat HCC.