Direct interaction of ß-catenin with nuclear ESM1 supports stemness of metastatic prostate cancer.

Wnt/ß-catenin signaling is frequently activated in advanced prostate cancer and contributes to therapy resistance and metastasis. However, activating mutations in the Wnt/ß-catenin pathway are not common in prostate cancer, suggesting alternative regulations may exist. Here, we report that the expression of endothelial cell-specific molecule 1 (ESM1), a secretory proteoglycan, is positively associated with prostate cancer stemness and progression by promoting Wnt/ß-catenin signaling. Elevated ESM1 expression correlates with poor overall survival and metastasis. Accumulation of nuclear ESM1, instead of cytosolic or secretory ESM1, supports prostate cancer stemness by interacting with the ARM domain of ß-catenin to stabilize ß-catenin-TCF4 complex and facilitate the transactivation of Wnt/ß-catenin signaling targets. Accordingly, activated ß-catenin in turn mediates the nuclear entry of ESM1. Our results establish the significance of mislocalized ESM1 in driving metastasis in prostate cancer by coordinating the Wnt/ß-catenin pathway, with implications for its potential use as a diagnostic or prognostic biomarker and as a candidate therapeutic target in prostate cancer.

MAOB expression correlates with a favourable prognosis in prostate cancer, and its genetic variants are associated with the metastasis of the disease.

Monoamine oxidase B (MAOB), a neurotransmitter-degrading enzyme, was reported to reveal conflicting roles in various cancers. However, the functional role of MAOB and impacts of its genetic variants on prostate cancer (PCa) is unknown. Herein, we genotyped four loci of MAOB single-nucleotide polymorphisms (SNPs), including rs1799836 (A/G), rs3027452 (G/A), rs6651806 (A/C) and rs6324 (G/A) in 702 PCa Taiwanese patients. We discovered that PCa patients carrying the MAOB rs6324 A-allele exhibited an increased risk of having a high initial prostate-specific antigen (iPSA) level (>10 ng/mL). Additionally, patients with the rs3027452 A-allele had a higher risk of developing distal metastasis, particularly in the subpopulation with high iPSA levels. In a subpopulation without postoperative biochemical recurrence, patients carrying the rs1799836 G-allele had a higher risk of developing lymph node metastasis and recurrence compared to those carrying the A-allele. Furthermore, genotype screening in PCa cell lines revealed that cells carrying the rs1799836 G-allele expressed lower MAOB levels than those carrying the A-allele. Functionally, overexpression and knockdown of MAOB in PCa cells respectively suppressed and enhanced cell motility and proliferation. In clinical observations, correlations of lower MAOB expression levels with higher Gleason scores, advanced clinical T stages, tumour metastasis, and poorer prognosis in PCa patients were noted. Our findings suggest that MAOB may act as a suppressor of PCa progression, and the rs3027452 and rs1799836 genetic variants of MAOB are linked to PCa metastasis within the Taiwanese population.

The oncogenic ADAMTS1-VCAN-EGFR cyclic axis drives anoikis resistance and invasion in renal cell carcinoma.

BACKGROUND: Metastasis, the leading cause of renal cell carcinoma (RCC) mortality, involves cancer cells resisting anoikis and invading. Until now, the role of the matrix metalloproteinase (MMP)-related enzyme, A disintegrin and metalloprotease with thrombospondin motifs 1 (ADAMTS1), in RCC anoikis regulation remains unclear. METHODS: The clinical significance of ADAMTS1 and its associated molecules in patients with RCC was investigated using data from the Gene Expression Omnibus (GEO) and TCGA datasets. Human phosphoreceptor tyrosine kinase (RTK) array, luciferase reporter assays, immunoprecipitation (IP) assays, western blotting, and real-time reverse-transcription quantitative polymerase chain reaction (RT-qPCR) were used to elucidate the underlying mechanisms of ADAMTS1. Functional assays, including anoikis resistance assays, invasion assays, and a Zebrafish xenotransplantation model, were conducted to assess the roles of ADAMTS1 in conferring resistance to anoikis in RCC. RESULTS: This study found elevated ADAMTS1 transcripts in RCC tissues that were correlated with a poor prognosis. ADAMTS1 manipulation significantly affected cell anoikis through the mitochondrial pathway in RCC cells. Human receptor tyrosine kinase (RTK) array screening identified that epidermal growth factor receptor (EGFR) activation was responsible for ADAMTS1-induced anoikis resistance and invasion. Further investigations revealed that enzymatically active ADAMTS1-induced versican V1 (VCAN V1) proteolysis led to EGFR transactivation, which in turn, through positive feedback, regulated ADAMTS1. Additionally, ADAMTS1 can form a complex with p53 to influence EGFR signaling. In vivo, VCAN or EGFR knockdown reversed ADAMTS1-induced prometastatic characteristics of RCC. A clinical analysis revealed a positive correlation between ADAMTS1 and VCAN or the EGFR and patients with RCC with high ADAMTS1 and VCAN expression had the worst prognoses. CONCLUSIONS: Our results collectively uncover a novel cyclic axis involving ADAMTS1-VCAN-EGFR, which significantly contributes to RCC invasion and resistance to anoikis, thus presenting a promising therapeutic target for RCC metastasis.

Genetic variants of dipeptidyl peptidase IV are linked to the clinicopathologic development of prostate cancer.

CD26/dipeptidyl peptidase IV (DPP4) is a multifunctional cell-surface glycoprotein widely found in many cell types, and a soluble form is present in body fluids. There is longstanding evidence indicating a tumour-promoting or -suppressive role of DPP4 in different cancer types. However, studies focusing on the impacts of genetic variants of DPP4 on cancers are very rare. Herein, we conducted a case-control study to evaluate whether single-nucleotide polymorphisms (SNPs) of DPP4 were associated with the risk or clinicopathologic development of prostate cancer (PCa). We genotyped four loci of DPP4 SNPs, including rs7608798 (A/G), rs3788979 (C/T), rs2268889 (T/C) and rs6741949 (G/C), using a TaqMan allelic discrimination assay in 704 PCa patients and 704 healthy controls. Our results showed that PCa patients with the DPP4 rs7608798 AG+GG genotype or rs2268889 TC+CC genotype had a higher risk of developing an advanced clinical primary tumour (cT) stage (adjusted odds ratio (AOR): 1.680, 95% confidence interval (CI): 1.062-2.659, p = 0.025; AOR: 1.693, 95% CI: 1.092-2.624, p = 0.018). Additionally, in The Cancer Genome Atlas (TCGA) database, we observed that lower DPP4 expression levels were correlated with higher Gleason scores, advanced cT and pathological stages, tumour metastasis, and shorter progression-free survival rates in PCa patients. Furthermore, overexpression of DPP4 suppressed migration/invasion of metastatic PC3 PCa cells. Our findings suggest that DPP4 levels may affect the progression of PCa, and the DPP4 rs7608798 and rs2268889 SNPs are associated with the clinicopathologic development of PCa in a Taiwanese population.

Functional variants of the chitinase 3-like 1 gene are associated with clinicopathologic outcomes and progression of prostate cancer.

Chitinase 3-like 1 (CHI3L1 or YKL40) is a secreted glycoprotein highly expressed in advanced stages of several cancer types, including prostate cancer (PCa). Impacts of genetic variants of CHI3L1 on PCa development have not yet been investigated. The most common well-studied genetic variations are single-nucleotide polymorphisms (SNPs). Therefore, the objective of this study was to explore associations of CHI3L1 SNPs with both the susceptibility to PCa and its clinicopathological development. Three promoter SNPs, rs6691378 (-1371, G>A), rs10399805 (-247, G>A) and rs4950928 (-131, C>G), and one non-synonymous SNP, rs880633 (+2950, T>C), were analysed using a TaqMan allelic discrimination assay for genotyping in a cohort of 701 PCa patients and 701 healthy controls. Results indicated that there were no significant associations of PCa susceptibility with these four CHI3L1 SNPs. However, among elderly PCa patients (aged >65 years), it was observed that polymorphic variants (GA + AA) of CHI3L1 rs6691378 and 10399805 were significantly linked to reduced risks of several clinicopathological characteristics, including a high Gleason grade, advanced pathologic T stage and tumour cell invasion. Moreover, analyses of The Cancer Genome Atlas database revealed that CHI3L1 expression levels were elevated in PCa tissues compared with normal tissues. Interestingly, higher CHI3L1 expression levels were found to be associated with longer progression-free survival rates in PCa patients. Our findings indicated that levels of CHI3L1 may influence the progression of PCa, and the rs6691378 and 10399805 SNP genetic variants of CHI3L1 are linked to the clinicopathological development of PCa within a Taiwanese population.

The RNA-binding protein KSRP aggravates malignant progression of clear cell renal cell carcinoma through transcriptional inhibition and post-transcriptional destabilization of the NEDD4L ubiquitin ligase.

BACKGROUND: KH-type splicing regulatory protein (KHSRP, also called KSRP), a versatile RNA-binding protein, plays a critical role in various physiological and pathological conditions through modulating gene expressions at multiple levels. However, the role of KSRP in clear cell renal cell carcinoma (ccRCC) remains poorly understood. METHODS: KSRP expression was detected by a ccRCC tissue microarray and evaluated by an in silico analysis. Cell loss-of-function and gain-of-function, colony-formation, anoikis, and transwell assays, and an orthotopic bioluminescent xenograft model were conducted to determine the functional role of KRSP in ccRCC progression. Micro (mi)RNA and complementary (c)DNA microarrays were used to identify downstream targets of KSRP. Western blotting, quantitative real-time polymerase chain reaction, and promoter- and 3-untranslated region (3'UTR)-luciferase reporter assays were employed to validate the underlying mechanisms of KSRP which aggravate progression of ccRCC. RESULTS: Our results showed that dysregulated high levels of KSRP were correlated with advanced clinical stages, larger tumor sizes, recurrence, and poor prognoses of ccRCC. Neural precursor cell-expressed developmentally downregulated 4 like (NEDD4L) was identified as a novel target of KSRP, which can reverse the protumorigenic and prometastatic characteristics as well as epithelial-mesenchymal transition (EMT) promotion by KSRP in vitro and in vivo. Molecular studies revealed that KSRP can decrease NEDD4L messenger (m)RNA stability via inducing mir-629-5p upregulation and directly targeting the AU-rich elements (AREs) of the 3'UTR. Moreover, KSRP was shown to transcriptionally suppress NEDD4L via inducing the transcriptional repressor, Wilm's tumor 1 (WT1). In the clinic, ccRCC samples revealed a positive correlation between KSRP and mesenchymal-related genes, and patients expressing high KSRP and low NEDD4L had the worst prognoses. CONCLUSION: The current findings unveil novel mechanisms of KSRP which promote malignant progression of ccRCC through transcriptional inhibition and post-transcriptional destabilization of NEDD4L transcripts. Targeting KSRP and its pathways may be a novel pharmaceutical intervention for ccRCC.

Genetic Polymorphisms of lncRNA LINC00673 as Predictors of Hepatocellular Carcinoma Progression in an Elderly Population.

Long noncoding (lnc)RNAs are reported to be key regulators of tumor progression, including hepatocellular carcinoma (HCC). The lncRNA long intergenic noncoding RNA 00673 (LINC00673) was indicated to play an important role in HCC progression, but the impacts of genetic variants (single-nucleotide polymorphisms, SNPs) of LINC00673 on HCC remain unclear. A TaqMan allelic discrimination assay was performed to analyze the genotypes of three tagging SNPs, viz., rs9914618 G > A, rs6501551 A > G, and rs11655237 C > T, of LINC00673 in 783 HCC patients and 1197 healthy subjects. Associations of functional SNPs of LINC00673 with HCC susceptibility and clinicopathologic variables were analyzed by logistic regression models. After stratification by confounding factor, we observed that elderly patients (≥60 years) with the LINC00673 rs9914618 A allele had an increased risk of developing HCC under a codominant model (p = 0.025) and dominant model (p = 0.047). Moreover, elderly patients carrying the GA + AA genotype of rs9914618 exhibited a higher risk of having lymph node metastasis compared to those who were homozygous for the major allele (p = 0.013). Genotype screening of rs9914618 in HCC cell lines showed that cells carrying the AA genotype expressed higher LINC00673 levels compared to the cells carrying the GG genotype. Further analyses of clinical datasets from the Cancer Genome Atlas (TCGA) showed that LINC00673 expressions were upregulated in HCC tissues compared to normal tissues, and were correlated with advanced clinical stages and poorer prognoses. In conclusions, our results suggested that the LINC00673 rs9914618 polymorphism may be a promising HCC biomarker, especially in elderly populations.

Proteomics-based identification of TMED9 is linked to vascular invasion and poor prognoses in patients with hepatocellular carcinoma.

BACKGROUND: Due to the difficulties in early diagnosing and treating hepatocellular carcinoma (HCC), prognoses for patients remained poor in the past decade. In this study, we established a screening model to discover novel prognostic biomarkers in HCC patients. METHODS: Candidate biomarkers were screened by liquid chromatography with tandem mass spectrometry (LC-MS/MS) analyses of five HCC normal (N)/tumor (T) paired tissues and preliminarily verified them through several in silico database analyses. Expression levels and functional roles of candidate biomarkers were respectively evaluated by immunohistochemical staining in N/T paired tissue (n = 120) and MTS, colony formation, and transwell migration/invasion assays in HCC cell lines. Associations of clinicopathological features and prognoses with candidate biomarkers in HCC patients were analyzed from GEO and TCGA datasets and our recruited cohort. RESULTS: We found that the transmembrane P24 trafficking protein 9 (TMED9) protein was elevated in HCC tissues according to a global proteomic analysis. Higher messenger (m)RNA and protein levels of TMED9 were observed in HCC tissues compared to normal liver tissues or pre-neoplastic lesions. The TMED9 mRNA expression level was significantly associated with an advanced stage and a poor prognosis of overall survival (OS, p = 0.00084) in HCC patients. Moreover, the TMED9 protein expression level was positively correlated with vascular invasion (p = 0.026), OS (p = 0.044), and disease-free survival (p = 0.015) in our recruited Taiwanese cohort. In vitro, manipulation of TMED9 expression in HCC cells significantly affected cell migratory, invasive, proliferative, and colony-forming abilities. CONCLUSIONS: Ours is the first work to identify an oncogenic role of TMED9 in HCC cells and may provide insights into the application of TMED9 as a novel predictor of clinical outcomes and a potential therapeutic target in patients with HCC.

PDK1 Inhibitor BX795 Improves Cisplatin and Radio-Efficacy in Oral Squamous Cell Carcinoma by Downregulating the PDK1/CD47/Akt-Mediated Glycolysis Signaling Pathway.

Background: Oral squamous cell carcinoma (OSCC) has a high prevalence and predicted global mortality rate of 67.1%, necessitating better therapeutic strategies. Moreover, the recurrence and resistance of OSCC after chemo/radioresistance remains a major bottleneck for its effective treatment. Molecular targeting is one of the new therapeutic approaches to target cancer. Among a plethora of targetable signaling molecules, PDK1 is currently rising as a potential target for cancer therapy. Its aberrant expression in many malignancies is observed associated with glycolytic re-programming and chemo/radioresistance. Methods: Furthermore, to better understand the role of PDK1 in OSCC, we analyzed tissue samples from 62 patients with OSCC for PDK1 expression. Combining in silico and in vitro analysis approaches, we determined the important association between PDK1/CD47/LDHA expression in OSCC. Next, we analyzed the effect of PDK1 expression and its connection with OSCC orosphere generation and maintenance, as well as the effect of the combination of the PDK1 inhibitor BX795, cisplatin and radiotherapy in targeting it. Results: Immunohistochemical analysis revealed that higher PDK1 expression is associated with a poor prognosis in OSCC. The immunoprecipitation assay indicated PDK1/CD47 binding. PDK1 ligation significantly impaired OSCC orosphere formation and downregulated Sox2, Oct4, and CD133 expression. The combination of BX795 and cisplatin markedly reduced in OSCC cell's epithelial-mesenchymal transition, implying its synergistic effect. p-PDK1, CD47, Akt, PFKP, PDK3 and LDHA protein expression were significantly reduced, with the strongest inhibition in the combination group. Chemo/radiotherapy together with abrogation of PDK1 inhibits the oncogenic (Akt/CD47) and glycolytic (LDHA/PFKP/PDK3) signaling and, enhanced or sensitizes OSCC to the anticancer drug effect through inducing apoptosis and DNA damage together with metabolic reprogramming. Conclusions: Therefore, the results from our current study may serve as a basis for developing new therapeutic strategies against chemo/radioresistant OSCC.

Melatonin-triggered post-transcriptional and post-translational modifications of ADAMTS1 coordinately retard tumorigenesis and metastasis of renal cell carcinoma.

A disintegrin and metalloprotease with thrombospondin motifs (ADAMTS) family are widely implicated in tissue remodeling events manifested in cancer development. ADAMTS1, the most fully characterized ADAMTS, plays conflicting roles in different cancer types; however, the role of ADAMTS1 in renal cell carcinoma (RCC) remains unclear. Herein, we found that ADAMTS1 is highly expressed in RCC tissues compared to normal renal tissues, and its expression was correlated with an advanced stage and a poor prognosis of RCC patients. In vitro, we observed higher expression of ADAMTS1 in metastatic (m)RCC cells compared to primary cells, and manipulation of ADAMTS1 expression affected cell invasion and clonogenicity. Results from protease array showed that ADAMTS1 is modulated by melatonin through mechanisms independent of the MT1 receptor in mRCC cells, and overexpression of ADAMTS1 relieved the invasion/clonogenicity and growth/metastasis inhibition imposed by melatonin treatment in vitro and in an orthotopic xenograft model. The human microRNA (miR) OneArray showed that miR-181d and miR-let-7f were induced by melatonin and, respectively, targeted the 3'-UTR and non-3'-UTR of ADAMTS1 to suppress its expression and mRCC invasive ability. Clinically, RCC patients with high levels of miR-181d or miR-let-7f and a low level of ADAMTS1 had the most favorable prognoses. In addition, ubiquitin/proteasome-mediated degradation of ADAMTS1 can also be triggered by melatonin. Together, our study indicates that ADAMTS1 may be a useful biomarker for predicting RCC progression. The novel convergence between melatonin and ADAMTS1 post-transcriptional and post-translational regulation provides new insights into the role of melatonin-induced molecular regulation in suppressing RCC progression.

Monoamine Oxidase B Expression Correlates with a Poor Prognosis in Colorectal Cancer Patients and Is Significantly Associated with Epithelial-to-Mesenchymal Transition-Related Gene Signatures.

Monoamine oxidases (MAOs) including MAOA and MAOB are enzymes located on the outer membranes of mitochondria, which are responsible for catalyzing monoamine oxidation. Recently, increased level of MAOs were shown in several cancer types. However, possible roles of MAOs have not yet been elucidated in the progression and prognosis of colorectal carcinoma (CRC). We therefore analyzed the importance of MAOs in CRC by an in silico analysis and tissue microarrays. Several independent cohorts indicated that high expression of MAOB, but not MAOA, was correlated with a worse disease stage and poorer survival. In total, 203 colorectal adenocarcinoma cases underwent immunohistochemical staining of MAOs, and associations with clinicopathological parameters and patient outcomes were evaluated. We found that MAOB is highly expressed in CRC tissues compared to normal colorectal tissues, and its expression was significantly correlated with a higher recurrence rate and a poor prognosis. Moreover, according to the univariate and multivariate analyses, we found that MAOB could be an independent prognostic factor for overall survival and disease-free survival, and its prognostic value was better than T and N stage. Furthermore, significant positive and negative correlations of MAOB with mesenchymal-type and epithelial-type gene expressions were observed in CRC tissues. According to the highlighted characteristics of MAOB in CRC, MAOB can be used as a novel indicator to predict the progression and prognosis of CRC patients.

Associations of TIMP-3 Genetic Polymorphisms with EGFR Statuses and Cancer Clinicopathologic Development in Lung Adenocarcinoma Patients.

Lung adenocarcinoma (LADC) is a major subtype of lung cancer, particularly among populations of East Asia. The epidermal growth factor receptor (EGFR) is the most frequently mutated oncogene promoting LADC progression and can serve as a therapeutic target in LADC. The tissue inhibitor of metalloproteinases (TIMP)-3 is a major regulator of extracellular matrix turnover via targeting of matrix metalloproteinases (MMPs), and thus, plays a critical role in tumor development and progression. The purpose of this study was to investigate potential associations among TIMP-3 genetic polymorphisms, EGFR statuses, and cancer clinicopathologic development in patients with LADC. In this study, 277 LADC patients with different EGFR statuses were recruited to dissect the allelic discrimination of TIMP-3 -1296 T>C (rs9619311), TIMP3 249T>C (rs9862), and TIMP3 261C>T (rs11547635) polymorphisms using a TaqMan allelic discrimination assay. Our data showed that compared to those LADC patients with wild-type CC homozygotes of TIMP-3 rs9862, patients harboring TT homozygotes of rs9862 were at a higher risk of developing mutant EGFR (adjusted odds ratio (AOR) = 2.530; 95% confidence interval (CI): 1.230-5.205; p = 0.012), particularly the EGFR L858R point mutation (AOR = 2.975; 95% CI: 1.182-7.488; p = 0.021). Moreover, we observed that TIMP-3 TT homozygotes of rs9862 were correlated with the incidence of EGFR mutations in patients with a smoking habit (p = 0.045). Within male patients harboring a mutant EGFR, TIMP-3 rs9862 T (CT+TT) allele carriers were at higher risk of developing an advanced stage (p = 0.025) and lymph node metastasis (p = 0.043). Further analyses of clinical datasets revealed correlations of TIMP-3 expression with a favorable prognosis in patients with LADC. In conclusion, the data suggest that TIMP-3 rs9862 polymorphisms may contribute to identify subgroups of lung cancer patients at high risk for tumor progression, among carriers of LADC-bearing mutant EGFR.

Penfluridol triggers cytoprotective autophagy and cellular apoptosis through ROS induction and activation of the PP2A-modulated MAPK pathway in acute myeloid leukemia with different FLT3 statuses.

BACKGROUND: Chemotherapy is the main treatment for acute myeloid leukemia (AML), but the cure rates for AML patients remain low, and the notorious adverse effects of chemotherapeutic drugs drastically reduce the life quality of patients. Penfluridol, a long-acting oral antipsychotic drug, has an outstanding safety record and exerts oncostatic effects on various solid tumors. Until now, the effect of penfluridol on AML remains unknown. METHODS: AML cell lines harboring wild-type (WT) Fms-like tyrosine kinase 3 (FLT3) and internal tandem duplication (ITD)-mutated FLT3 were used to evaluate the cytotoxic effects of penfluridol by an MTS assay. A flow cytometric analysis and immunofluorescence staining were employed to determine the cell-death phenotype, cell cycle profile, and reactive oxygen species (ROS) and acidic vesicular organelle (AVO) formation. Western blotting and chemical inhibitors were used to explore the underlying mechanisms involved in penfluridol-mediated cell death. RESULTS: We observed that penfluridol concentration-dependently suppressed the cell viability of AML cells with FLT3-WT (HL-60 and U937) and FLT3-ITD (MV4-11). We found that penfluridol treatment not only induced apoptosis as evidenced by increases of nuclear fragmentation, the sub-G1 populations, poly (ADP ribose) polymerase (PARP) cleavage, and caspase-3 activation, but also triggered autophagic responses, such as the light chain 3 (LC3) turnover and AVO formation. Interestingly, blocking autophagy by the pharmacological inhibitors, 3-methyladenine and chloroquine, dramatically enhanced penfluridol-induced apoptosis, indicating the cytoprotective role of autophagy in penfluridol-treated AML cells. Mechanistically, penfluridol-induced apoptosis occurred through activating protein phosphatase 2A (PP2A) to suppress Akt and mitogen-activated protein kinase (MAPK) activities. Moreover, penfluridol's augmentation of intracellular ROS levels was critical for the penfluridol-induced autophagic response. In the clinic, we observed that patients with AML expressing high PP2A had favorable prognoses. CONCLUSIONS: These findings provide a rationale for penfluridol being used as a PP2A activator for AML treatment, and the combination of penfluridol with an autophagy inhibitor may be a novel strategy for AML harboring FLT3-WT and FLT3-ITD.

Quercetin suppresses the metastatic ability of lung cancer through inhibiting Snail-dependent Akt activation and Snail-independent ADAM9 expression pathways.

Metastasis is the major cause of death from lung cancer. Quercetin, a widely distributed bioflavonoid, is well known to induce growth inhibition in a variety of human cancer cells, but how it affects lung cancer cell invasion and metastasis is unclear. Herein, we found that quercetin inhibited the migration/invasion of non-small cell lung cancer (NSCLC) cell lines and bone metastasis in an orthotopic A549 xenograft model by suppressing the Snail-mediated epithelial-to-mesenchymal transition (EMT). Moreover, survival times of animals were also prolonged after quercetin treatment. Mechanistic investigations found that quercetin suppressed Snail-dependent Akt activation by upregulating maspin and Snail-independent a disintegrin and metalloproteinase (ADAM) 9 expression pathways to modulate the invasive ability of NSCLC cells. In clinical samples, we observed that patients with Snailhigh/p-Akthigh tumors had the shortest survival times. In addition, a lower survival rate was also found in ADAM9high patients than in ADAM9low patients. Overall, our results provide new insights into the role of quercetin-induced molecular regulation in suppressing NSCLC metastasis and suggest that quercetin has potential therapeutic applications for metastatic NSCLC.

Leukocyte Cell-Derived Chemotaxin 2 Retards Non-Small Cell Lung Cancer Progression Through Antagonizing MET and EGFR Activities.

BACKGROUND/AIMS: Epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor (TKI) therapy is a clinical option for non-small cell lung cancer (NSCLC) harboring activating EGFR mutations or for cancer with wild-type (WT) EGFR when chemotherapy has failed. MET receptor activation or MET gene amplification was reported to be a major mechanism of acquired resistance to EGFR-TKI therapy in NSCLC cells. Leukocyte cell-derived chemotaxin 2 (LECT2) is a multifunctional cytokine that was shown to suppress metastasis of hepatocellular carcinoma via inhibiting MET activity. Until now, the biological function responsible for LECT2's action in human NSCLC remains unclear. METHODS: LECT2-knockout (KO) mice and NOD/SCID/IL2rgnull (NSG) mice were respectively used to investigate the effects of LECT2 on the tumorigenicity and metastasis of murine (Lewis lung carcinoma, LLC) and human (HCC827) lung cancer cells. The effect of LECT2 on in vitro cell proliferation was evaluated, using MTS and colony formation assays. The effect of LECT2 on cell motility was evaluated using transwell migration and invasion assays. An enzyme-linked immunosorbent assay was performed to detect secreted LECT2 in plasma and media. Co-immunoprecipitation and Western blot assays were used to investigate the underlying mechanisms of LECT2 in NSCLC cells. RESULTS: Compared to WT mice, mice with LECT2 deletion exhibited enhanced growth and metastasis of LLC cells, and survival times decreased in LLC-implanted mice. Overexpression of LECT2 in orthotopic human HCC827 xenografts in NSG mice resulted in significant inhibition of tumor growth and metastasis. In vitro, overexpression of LECT2 or treatment with a recombinant LECT2 protein impaired the colony-forming ability and motility of NSCLC cells (HCC827 and PC9) harboring high levels of activated EGFR and MET. Mechanistic investigations found that LECT2 bound to MET and EGFR to antagonize their activation and further suppress their common downstream pathways: phosphatidylinositol 3-kinase/Akt and extracellular signal-regulated kinase. CONCLUSION: EGFR-MET signaling is critical for aggressive behaviors of NSCLC and is recognized as a therapeutic target for NSCLC especially for patients with acquired resistance to EGFR-TKI therapy. Our findings demonstrate, for the first time, that LECT2 functions as a suppressor of the progression of NSCLC by targeting EGFR-MET signaling.

Stimulation of Fas/FasL-mediated apoptosis by luteolin through enhancement of histone H3 acetylation and c-Jun activation in HL-60 leukemia cells.

Luteolin (3',4',5,7-tetrahydroxyflavone), which exists in fruits, vegetables, and medicinal herbs, is used in Chinese traditional medicine for treating various diseases, such as hypertension, inflammatory disorders, and cancer. However, the gene-regulatory role of luteolin in cancer prevention and therapy has not been clarified. Herein, we demonstrated that treatment with luteolin resulted in a significant decrease in the viability of human leukemia cells. In the present study, by evaluating fragmentation of DNA and poly (ADP-ribose) polymerase (PARP), we found that luteolin was able to induce PARP cleavage and nuclear fragmentation as well as an increase in the sub-G0 /G1 fraction. In addition, luteolin also induced Fas and Fas ligand (FasL) expressions and subsequent activation of caspases-8 and -3, which can trigger the extrinsic apoptosis pathway, while knocking down Fas-associated protein with death domain (FADD) prevented luteolin-induced PARP cleavage. Immunoblot and chromatin immunoprecipitation (ChIP) analyses revealed that luteolin increased acetylation of histone H3, which is involved in the upregulation of Fas and FasL. Moreover, both the extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) pathways are involved in luteolin-induced histone H3 acetylation. Finally, luteolin also activated the c-Jun signaling pathway, which contributes to FasL, but not Fas, gene expression and downregulation of c-Jun expression by small interfering RNA transfection which resulted in a significant decrease in luteolin-induced PARP cleavage. Thus, our results demonstrate that luteolin induced apoptosis of HL-60 cells, and this was associated with c-Jun activation and histone H3 acetylation-mediated Fas/FasL expressions.

Associations of genetic variations of the endothelial nitric oxide synthase gene and environmental carcinogens with oral cancer susceptibility and development.

Oral cancer is a major head and neck cancer that is reported to be causally associated with genetic factors and environmental carcinogens. Endothelial nitric oxide synthase (eNOS) was reported to modulate carcinogenesis and progression through nitric oxide (NO) production. Genetic polymorphisms in the eNOS gene can regulate its transcription and further mediate NO production. The purpose of this study was to explore the influences of eNOS gene polymorphisms combined with environmental carcinogens on the predisposition for oral cancer. Two single-nucleotide polymorphisms (SNPs) of the eNOS gene, -786 T > C (rs2070744) and 894G > T (rs1799983), were genotyped in 1200 controls and 1044 patients with oral cancer using a TaqMan-based real-time polymerase chain reaction (PCR). We found that patients who carried the -786 T > C TC genotype were at higher risk for developing an advanced clinical stage (stage III/IV) compared to those with the -786 T > C TT genotype; however, there was no significant association of the two individual SNPs with oral cancer between patients and the control group. According to behavioral exposure to environmental carcinogens, the presence of these two eNOS SNPs combined with tobacco use and/or betel quid chewing profoundly enhanced the risk of oral cancer. Moreover, carriers with the betel quid-chewing habit who had haplotypes of the two eNOS SNPs more easily developed oral cancer. These results indicated an involvement of -786 T > C polymorphisms in the progression of oral cancer and support the interaction between eNOS gene polymorphisms and environmental carcinogens as a predisposing factor of oral carcinogenesis.

Melatonin attenuates osteosarcoma cell invasion by suppression of C-C motif chemokine ligand 24 through inhibition of the c-Jun N-terminal kinase pathway.

Osteosarcoma, with its high metastatic potential, is the most prevalent malignant bone tumor in children and adolescents. Melatonin possesses multiple tumor-suppressing properties for a myriad of tumors, but little is known about the effects of melatonin on osteosarcoma metastasis. In this study, we demonstrated that melatonin elicited very low cytotoxicity and significantly inhibited cellular motility, migration, and invasion in human osteosarcoma U2OS and HOS cells. Moreover, using RNA sequencing technology, we revealed that melatonin repressed C-C motif chemokine ligand 24 (CCL24) gene expression in U2OS cells. Manipulation of CCL24 levels influenced the motility of osteosarcoma cells as cell migration and invasion were enhanced by the addition of recombinant human CCL24 and attenuated by the silencing of CCL24. Moreover, melatonin increased and decreased the activation of extracellular signal-regulated kinase (ERK) 1/2 and c-Jun N-terminal kinase (JNK) 1/2, respectively, in a dose-dependent manner in U2OS and HOS cells while exerting no evident influence on the level and activation of p38, Akt, FAK, steroid receptor coactivator, or Raf. In further functional experiments, the use of JNK inhibitors (SP600125 and DN-JNK) confirmed that the pharmaceutic inhibition of JNK augmented the melatonin-mediated CCL24 suppression and migration of U2OS cells. Overall, our results revealed that melatonin attenuated chemokine CCL24 levels through inhibition of the JNK pathway to hinder human osteosarcoma cell invasion, thereby highlighting the therapeutic potential of melatonin for osteosarcoma metastasis.

Dopamine receptor D2 genetic variations is associated with the risk and clinicopathological variables of urothelial cell carcinoma in a Taiwanese population.

Dopamine receptor D2 (DRD2) is overexpressed in several kinds of cancers and was correlated with the prognosis of these cancers. Polymorphisms within the DRD2 gene were shown to be associated with lung and colon cancers. The purpose of this study was to explore effects of DRD2 gene polymorphisms on the susceptibility to and clinicopathological characteristics of urothelial cell carcinoma (UCC). In total, 369 patients diagnosed with UCC and 738 healthy controls were enrolled to analyze DRD2 genotypes at four loci (rs1799732, -141C>del; rs1079597, TaqIB; rs6277, 957C>T; and rs1800497, TaqIA) using a TaqMan-based real-time polymerase chain reaction (PCR). We found a significantly lower risk for UCC in individuals with the DRD2 rs6277 CT genotype compared to those with the wild-type CC genotype (adjusted odds ratio (AOR)=0.405, 95% confidence interval (CI): 0.196~0.837, p=0.015). In 124 younger patients (aged < 65 years) of the recruited UCC cohort, patients who carried at least one T allele of DRD2 rs1800497 were at higher risk (AOR=2.270, 95% CI: 1.060~4.860, p=0.033) of developing an invasive stage (pT2~pT4). In 128 female patients of the recruited UCC cohort, patients who carried at least one deletion allele of DRD2 rs1799732 showed a higher incidence of having an invasive stage (AOR=2.585, 95% CI: 1.066~6.264, p=0.032) and a large tumor (AOR=2.778, 95% CI: 1.146~6.735, p=0.021). Further analyses of The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) datasets revealed correlations of the expression of DRD2 with an invasive tumor, tumor metastasis, and the lower survival rate in patients with UCC. Our findings suggest that DRD2 levels might affect the progression of UCC, and the polymorphisms rs6277, rs1800497, and rs1799732 of DRD2 are probably associated with the susceptibility and clinicopathologic development of UCC in a Taiwanese population.

Niclosamide, an oral antihelmintic drug, exhibits antimetastatic activity in hepatocellular carcinoma cells through downregulating twist-mediated CD10 expression.

Hepatocellular carcinoma (HCC) is one of the most common malignancies in the world, especially, in eastern Asia, and its prognosis is poor once metastasis occurs. Niclosamide, a US Food and Drug Administration-approved antihelmintic drug, was shown to inhibit the growth of various cancers including HCC, but the effect of niclosamide on cell motility and the underlying mechanism have not yet been completely defined. The present study demonstrated that niclosamide, at 0-40 nM, concentration-dependently inhibited wound closure and the migratory/invasive capacities of human Huh7 and SK-Hep-1 HCC cells without exhibiting cytotoxicity. A protease array analysis showed that CD10 was dramatically downregulated in Huh7 cells after niclosamide treatment. Western blot and flow cytometric assays further demonstrated that CD10 expression was concentration-dependently downregulated in Huh7 and SK-Hep-1 cells after niclosamide treatment. Mechanistic investigations found that niclosamide suppressed Twist-mediated CD10 transactivation. Moreover, knockdown of CD10 expression by CD10 small interfering RNA in HCC cells suppressed cell migratory/invasive abilities and overexpression of CD10 relieved the migration inhibition induced by niclosamide. Taken together, our results indicated that niclosamide could be a potential agent for inhibiting metastasis of HCC, and CD10 is an important target of niclosamide for suppressing the motility of HCC cells.