Asparagine synthetase regulates lung-cancer metastasis by stabilizing the ß-catenin complex and modulating mitochondrial response.

The availability of asparagine is the limitation of cell growth and metastasis. Asparagine synthetase (ASNS) was an essential enzyme for endogenous asparagine products. In our study, ASNS-induced asparagine products were essential to maintain tumor growth and colony formations in vitro. But mutated ASNS which defected endogenous asparagine products still upregulated cell invasiveness, which indicated that ASNS promoted invasiveness by alternative pathways. Mechanically, ASNS modulated Wnt signal transduction by promoting GSK3ß phosphorylation on ser9 and stabilizing the ß-catenin complex, as result, ASNS could promote more ß-catenin translocation into nucleus independent of endogenous asparagine. At the same time, ASNS modulated mitochondrial response to Wnt stimuli with increased mitochondrial potential and membrane fusion. In summary, ASNS promoted metastasis depending on Wnt pathway and mitochondrial functions even without endogenous asparagine products.

DIAPH3 promotes pancreatic cancer progression by activating selenoprotein TrxR1-mediated antioxidant effects.

Pancreatic cancer is a highly malignant tumour of the digestive tract which is difficult to diagnose and treat. Approximately 90% of cases arise from ductal adenocarcinoma of the glandular epithelium. The morbidity and mortality of the disease have increased significantly in recent years. Its 5-year survival rate is <1% and has one of the worst prognoses amongst malignant tumours. Pancreatic cancer has a low rate of early-stage diagnosis, high surgical mortality and low cure rate. Selenium compounds produced by selenoamino acid metabolism may promote a large amount of oxidative stress and subsequent unfolded reactions and endoplasmic reticulum stress by consuming the NADPH in cells, and eventually lead to apoptosis, necrosis or necrotic cell death. In this study, we first identified DIAPH3 as a highly expressed protein in the tissues of patients with pancreatic cancer, and confirmed that DIAPH3 promoted the proliferation, anchorage-independent growth and invasion of pancreatic cancer cells using overexpression and interference experiments. Secondly, bioinformatics data mining showed that the potential proteins interacted with DIAPH3 were involved in selenoamino acid metabolism regulation. Selenium may be incorporated into selenoprotein synthesis such as TrxR1 and GPX4, which direct reduction of hydroperoxides or resist ferroptosis, respectively. Our following validation confirmed that DIAPH3 promoted selenium content and interacted with the selenoprotein RPL6, a ribosome protein subunit involved in selenoamino acid metabolism. In addition, we verified that DIAPH3 could down-regulate cellular ROS level via up-regulating TrxR1 expression. Finally, nude mice xenograft model experimental results demonstrate DIAPH3 knock down could decrease tumour growth and TrxR1 expression and ROS levels in vivo. Collectively, our observations indicate DIAPH3 could promote pancreatic cancer progression by activating selenoprotein TrxR1-mediated antioxidant effects.

m6A Reader YTHDC2 Promotes Radiotherapy Resistance of Nasopharyngeal Carcinoma via Activating IGF1R/AKT/S6 Signaling Axis.

N6-methyladenosine (m6A) modification has been reported as a critical regulator of gene transcript expression. Although m6A modification plays important roles in tumor development, its role in therapeutic resistance remains unknown. In this study, we aimed to examine the expression level of m6A-modification related proteins and elucidate the effect of m6A-related proteins on radiation response in nasopharyngeal carcinoma (NPC). Among the genes that participated in m6A modification, YTHDC2, a m6A reader, was found to be consistently highly expressed in radioresistant NPC cells. Knocking down of YTHDC2 expression in radioresistant NPC cells improved the therapeutic effect of radiotherapy in vitro and in vivo, whereas overexpression of YTHDC2 in radiosensitive NPC cells exerted an opposite effect. Bioinformatics and mechanistic studies revealed that YTHDC2 could physically bound to insulin-like growth factor 1 receptor (IGF1R) messenger RNA and promoted translation initiation of IGF1R mRNA, which in turn activated the IGF1R-AKT/S6 signaling pathway. Thus, the present study suggests that YTHDC2 promotes radiotherapy resistance of NPC cells by activating the IGF1R/ATK/S6 signaling axis and may serve as a potential therapeutic target in radiosensitization of NPC cells.

Downregulation of Fat Mass and Obesity Associated (FTO) Promotes the Progression of Intrahepatic Cholangiocarcinoma.

Intrahepatic cholangiocarcinoma (ICC) ranks as the second most malignant type of primary liver cancer with a high degree of incidence and a very poor prognosis. Fat mass and obesity-associated protein (FTO) functions as an eraser of the RNA m6A modification, but its roles in ICC tumorigenesis and development remain unknown. We showed here that the protein level of FTO was downregulated in clinical ICC samples and cell lines and that FTO expression was inversely correlated with the expression of CA19-9 and micro-vessel density (MVD). A Kaplan-Meier survival analysis showed that a low expression of FTO predicted poor prognosis in ICC. in vitro, decreased endogenous expression of FTO obviously reduced apoptosis of ICC cells. Moreover, FTO suppressed the anchorage-independent growth and mobility of ICC cells. Through mining the database, FTO was found to regulate the integrin signaling pathway, inflammation signaling pathway, epidermal growth factor receptor (EGFR) signaling pathway, angiogenesis, and the pyrimidine metabolism pathway. RNA decay assay showed that oncogene TEAD2 mRNA stability was impaired by FTO. In addition, the overexpression of FTO suppressed tumor growth in vivo. In conclusion, our study demonstrated the critical roles of FTO in ICC.

PRMT1 Promoted HCC Growth and Metastasis In Vitro and In Vivo via Activating the STAT3 Signalling Pathway.

BACKGROUND/AIMS: Although it has been widely accepted that protein arginine methyltransferase 1 (PRMT1) is a cancer-promoting gene in various cancers, the mechanism of PRMT1 in hepatocellular carcinoma (HCC) requires more exploration. This study aimed to investigate the role of PRMT1 in HCC growth and metastasis. METHODS: We compared PRMT1 expression and clinicopathological characteristics using paired HCC and adjacent noncancerous liver tissues from 210 patients and immunohistochemistry analyses. Cell proliferation, colony formation and migration were determined in HCC cell lines with PRMT1 overexpression or downregulation through MTT, crystal violet and Boyden chamber assays. Tumour growth was monitored in a xenograft model, and intrahepatic metastasis models were established. RESULTS: PRMT1 expression was greatly increased in clinical HCC samples and strongly associated with poor prognosis and recurrence; PRMT1 expression was also positively correlated with microvascular invasion (P = 0.024), tumour differentiation (P = 0.014), tumour size (P = 0.002), and portal vein tumour thrombus (PVTT) (P = 0.028). Cell proliferation, colony formation and migration in vitro were enhanced by PRMT1 upregulation and decreased by PRMT1 downregulation in HCC cell lines. Moreover, low PRMT1 expression resulted in slow tumour growth and decreased tumour weight in vivo, as well as tumour metastasis. These phenotypes were associated with STAT3 signalling pathway activation. Cryptotanshinone, a STAT3 inhibitor, inhibited STAT3 phosphorylation and reversed the HCC phenotype of PRMT1 expression. CONCLUSIONS: We revealed a significant role for PRMT1 in HCC progression and metastasis in vitro and in vivo via STAT3 signalling pathway activation. PRMT1 may be a potential novel prognostic biomarker and new therapeutic target for HCC.

Chemerin suppresses hepatocellular carcinoma metastasis through CMKLR1-PTEN-Akt axis.

BACKGROUND: Chemerin, a known chemoattractant, participates in multiple biological events. However, its role in cancer remains largely unknown. METHODS: Chemerin expression was evaluated by real-time PCR, western blot and immunohistochemistry. Forced expression, RNAi, immunoprecipitation, etc. were used in function and mechanism study. Mouse models of extrahepatic and intrahepatic metastasis were employed to evaluate the therapeutic potential of chemerin. RESULTS: Chemerin expression was significantly downregulated in hepatocellular carcinoma, and associated with poor prognosis of HCC patients. Forced expression of chemerin inhibited in vitro migration, invasion and in vivo metastasis of HCC cells. Administration of chemerin effectively suppressed extrahepatic and intrahepatic metastases of HCC cells, resulting in prolonged survival of tumour-bearing nude mice. Chemerin upregulated expression and phosphatase activity of PTEN by interfering with PTEN-CMKLR1 interaction, leading to weakened ubiquitination of PTEN and decreased p-Akt (Ser473) level, which was responsible for suppressed migration, invasion and metastasis of HCC cells. Positive correlation between chemerin and PTEN, and reverse correlation between chemerin and p-Akt (Ser473) were also observed in HCC clinical samples and intrahepatic mouse model in vivo. CONCLUSIONS: Our study has revealed the suppressive role and therapeutic potential of chemerin in HCC metastasis, providing both a prognostic marker and drug candidate for HCC.

hPCL3s Promotes Hepatocellular Carcinoma Metastasis by Activating ß-Catenin Signaling.

Two isoforms of human Polycomb-like protein 3 (hPCL3) have been reported as components of the nuclear Polycomb repressive complex 2 (PRC2), with the short isoform (hPCL3s) showing a dominant cytoplasmic localization. The function of cytoplasmic hPCL3s has, however, not been addressed. In this study, we report that hPCL3s is upregulated in clinical hepatocellular carcinoma (HCC) samples and its expression correlated with HCC clinical features. hPCL3s positively regulated the migration, invasion, and metastasis of HCC cells. hPCL3s interacted with components of the cytoplasmic ß-catenin destruction complex, inhibited ß-catenin degradation, and activated ß-catenin/T-cell factor signaling. Downstream of the ß-catenin cascade, IL6 mediated the motility-promoting functions of hPCL3s. Forced expression of hPCL3s in the liver of a HCC mouse model promoted tumorigenesis and metastasis. Taken together, these data show that hPCL3s promotes the metastasis of HCC by activating the ß-catenin/IL6 pathway.Significance: hPCL3s has an oncogenic role in hepatocellular carcinoma by activating the ß-catenin/IL6 signaling axis to promote metastasis. Cancer Res; 78(10); 2536-49. ©2018 AACR.

Cilia loss sensitizes cells to transformation by activating the mevalonate pathway.

Although cilia loss and cell transformation are frequently observed in the early stage of tumorigenesis, the roles of cilia in cell transformation are unknown. In this study, disrupted ciliogenesis was observed in cancer cells and pancreatic cancer tissues, which facilitated oncogene-induced transformation of normal pancreatic cells (HPDE6C7) and NIH3T3 cells through activating the mevalonate (MVA) pathway. Disruption of ciliogenesis up-regulated MVA enzymes through ß catenin-T cell factor (TCF) signaling, which synchronized with sterol regulatory element binding transcription factor 2 (SREBP2), and the regulation of MVA by ß-catenin-TCF signaling was recapitulated in a mouse model of pancreatic ductal adenocarcinoma (PDAC) and human PDAC samples. Moreover, disruption of ciliogenesis by depleting Tg737 dramatically promoted tumorigenesis in the PDAC mouse model, driven by KrasG12D , which was inhibited by statin, an inhibitor of the MVA pathway. Collectively, this study emphasizes the crucial roles of cilia in governing the early steps of the transformation by activating the MVA pathway, suggesting that statin has therapeutic potential for pancreatic cancer treatment.

Iron overload in hereditary tyrosinemia type 1 induces liver injury through the Sp1/Tfr2/hepcidin axis.

BACKGROUND & AIMS: Iron is an essential metal for fundamental metabolic processes, but little is known regarding the involvement of iron in other nutritional disorders. In the present study, we investigated disordered iron metabolism in a murine model of hereditary tyrosinemia type I (HT1), a disease of the tyrosine degradation pathway. METHODS: We analysed the status of iron accumulation following NTBC withdrawal from Fah(-/-) mice, a murine model for HT1. Liver histology and serum parameters were used to assess the extent of liver injury and iron deposition. To determine the physiological significance of iron accumulation, mice were subjected to a low-iron food intake to reduce the iron accumulation. Mechanistic studies were performed on tissues and cells using immunoblotting, qRT-PCR, adenovirus transfection and other assays. RESULTS: Severe iron overload was observed in the murine model of HT1 with dramatically elevated hepatic and serum iron levels. Mechanistic studies revealed that downregulation and dysfunction of Tfr2 decreased hepcidin, leading to iron overload. The Fah(-/-) hepatocytes lost the ability of transferrin-sensitive induction of hepcidin. Forced expression of Tfr2 in the murine liver reduced the iron accumulation. Moreover, transcription factor Sp1 was downregulated and identified as a new regulator of Tfr2 here. Additionally, low-iron food intake effectively reduced the iron deposits, protected the liver and prolonged the survival in these mice. CONCLUSIONS: Iron was severely overloaded in the HT1 mice via the Sp1/Tfr2/Hepcidin axis. The iron overload induced liver injury in the HT1 mice, and reduction of the iron accumulation ameliorated liver injury. LAY SUMMARY: Primary and secondary iron overload is an abnormal status affecting millions of people worldwide. Here, we reported severe iron overload in a murine model of HT1, a disease of the tyrosine degradation pathway, and elucidated the mechanistic basis and the physiological significance of iron overload in HT1. These studies are of general interest not only with respect to secondary iron-induced liver injury in HT1 but also are important to elucidate the crosstalk between the two metabolic pathways.

Sorafenib enriches epithelial cell adhesion molecule-positive tumor initiating cells and exacerbates a subtype of hepatocellular carcinoma through TSC2-AKT cascade.

UNLABELLED: Sorafenib is a specific adenosine triphosphate-competitive RAF inhibitor used as a first-line treatment of advanced hepatocellular carcinoma (HCC). However, the responses are variable, reflecting heterogeneity of the disease, while the resistance mechanism remains poorly understood. Here, we report that sorafenib treatment can exacerbate disease progression in both patient-derived xenografts and cell line-derived xenografts and that the therapeutic effect of the drug inversely covaries to the ratio of epithelial cell adhesion molecule-positive cells, which may be tumor initiating cells in HCC. The TSC2-AKT cascade mediates this sorafenib resistance. In response to sorafenib treatment, formation of the TSC1/2 complex is enhanced, causing increased phosphorylation of AKT, which contributes to up-regulation of "stemness"-related genes in epithelial cell adhesion molecule-positive cells and enhancement of tumorigenicity. The expression of TSC2 negatively correlated with prognosis in clinical sorafenib therapy. Furthermore, all-trans retinoic acid decreased AKT activity, reduced the epithelial cell adhesion molecule-positive cell population enriched by sorafenib, and potentiated the therapeutic effect of sorafenib in the patient-derived xenograft model. CONCLUSION: Our findings suggest that a subtype of HCC is not suitable for sorafenib therapy; this resistance to sorafenib can be predicted by the status of TSC2, and agents inducing differentiation of tumor initiating cells (e.g., all-trans retinoic acid) should improve the prognosis of this subtype of HCC.

RACK1 modulates NF-κB activation by interfering with the interaction between TRAF2 and the IKK complex.

The transcription factor NF-κB plays a pivotal role in innate immunity in response to a variety of stimuli, and the coordinated regulation of this pathway determines the proper host responses to extracellular signals. In this study, we identified RACK1 as a novel negative regulator of NF-κB signaling, NF-κB-mediated cytokine induction and inflammatory reactions. RACK1 physically associates with the IKK complex in a TNF-triggered manner. This interaction interferes with the recruitment of the IKK complex to TRAF2, which is a critical step for IKK phosphorylation and subsequent activation triggered by TNF. By modulating the interaction between TRAF2 and IKK, RACK1 regulates the levels of NF-κB activation in response to different intensities of stimuli. Our findings suggest that RACK1 plays an important role in controlling the sensitivity of TNF-triggered NF-κB signaling by regulating IKK activation and provide new insight into the negative regulation of inflammatory reactions.

EphB3 suppresses non-small-cell lung cancer metastasis via a PP2A/RACK1/Akt signalling complex.

Eph receptors are implicated in regulating the malignant progression of cancer. Here we find that despite overexpression of EphB3 in human non-small-cell lung cancer, as reported previously, the expression of its cognate ligands, either ephrin-B1 or ephrin-B2, is significantly downregulated, leading to reduced tyrosine phosphorylation of EphB3. Forced activation of EphB3 kinase in EphB3-overexpressing non-small-cell lung cancer cells inhibits cell migratory capability in vitro as well as metastatic seeding in vivo. Furthermore, we identify a novel EphB3-binding protein, the receptor for activated C-kinase 1, which mediates the assembly of a ternary signal complex comprising protein phosphatase 2A, Akt and itself in response to EphB3 activation, leading to reduced Akt phosphorylation and subsequent inhibition of cell migration. Our study reveals a novel tumour-suppressive signalling pathway associated with kinase-activated EphB3 in non-small-cell lung cancer, and provides a potential therapeutic strategy by activating EphB3 signalling, thus inhibiting tumour metastasis.

RACK1 promotes non-small-cell lung cancer tumorigenicity through activating sonic hedgehog signaling pathway.

Non-small-cell lung cancer (NSCLC) is a deadly disease due to lack of effective diagnosis biomarker and therapeutic target. Much effort has been made in defining gene defects in NSCLC, but its full molecular pathogenesis remains unexplored. Here, we found RACK1 (receptor of activated kinase 1) was elevated in most NSCLC, and its expression level correlated with key pathological characteristics including tumor differentiation, stage, and metastasis. In addition, RACK1 activated sonic hedgehog signaling pathway by interacting with and activating Smoothened to mediate Gli1-dependent transcription in NSCLC cells. And silencing RACK1 dramatically inhibited in vivo tumor growth and metastasis by blocking the sonic hedgehog signaling pathway. These results suggest that RACK1 represents a new promising diagnosis biomarker and therapeutic target for NSCLC.

RACK1 suppresses gastric tumorigenesis by stabilizing the ß-catenin destruction complex.

BACKGROUND & AIMS: Dysregulation of Wnt signaling has been involved in gastric tumorigenesis by mechanisms that are not fully understood. The receptor for activated protein kinase C (RACK1, GNB2L1) is involved in development of different tumor types, but its expression and function have not been investigated in gastric tumors. METHODS: We analyzed expression of RACK1 in gastric tumor samples and their matched normal tissues from 116 patients using immunohistochemistry. Effects of knockdown with small interfering RNAs or overexpression of RACK1 in gastric cancer cell lines were evaluated in cell growth and tumor xenograft. RACK1 signaling pathways were investigated in cells and zebrafish embryos using immunoblot, immunoprecipitation, microinjection, and in situ hybridization assays. RESULTS: Expression of RACK1 was reduced in gastric tumor samples and correlated with depth of tumor infiltration and poor differentiation. Knockdown of RACK1 in gastric cancer cells accelerated their anchorage-independent proliferation in soft agar, whereas overexpression of RACK1 reduced their tumorigenicity in nude mice. RACK1 formed a complex with glycogen synthase kinase Gsk3ß and Axin to promote the interaction between Gsk3ß and ß-catenin and thereby stabilized the ß-catenin destruction complex. On stimulation of Wnt3a, RACK1 repressed Wnt signaling by inhibiting recruitment of Axin by Dishevelled 2 (Dvl2). Moreover, there was an inverse correlation between expression of RACK1 and localization of ß-catenin to the cytoplasm/nucleus in human gastric tumor samples. CONCLUSIONS: RACK1 negatively regulates Wnt signaling pathway by stabilizing the ß-catenin destruction complex and act as a tumor suppressor in gastric cancer cells.

Sorafenib suppresses postsurgical recurrence and metastasis of hepatocellular carcinoma in an orthotopic mouse model.

UNLABELLED: Surgical resection is the first-line treatment for hepatocellular carcinoma (HCC) patients with well-preserved liver function. Nevertheless, the rate of postoperative recurrence at 5 years is as high as 70%, and this gravely jeopardizes the therapeutic outcome. Clearly, new approaches are needed for preventing the relapse of this deadly disease. Taking advantage of a luciferase-labeled orthotopic xenograft model of HCC, we examined the role of sorafenib, the first systemic drug approved for advanced HCC patients, in the prevention of HCC recurrence. We found that sorafenib suppressed the development of postsurgical intrahepatic recurrence and abdominal metastasis and consequently led to prolonged postoperative survival of mice in this model. Furthermore, hyperactivity of extracellular signal-regulated kinase signaling caused by elevated levels of growth factors associated with postoperative liver regeneration enhanced the sensitivity of HCC cells to sorafenib; this provides a plausible explanation for the observation that recurrent tumors are more responsive to growth inhibition by sorafenib. CONCLUSION: Our results strongly suggest that by effectively reducing postoperative recurrence, sorafenib has a potential application in early-stage HCC patients who have undergone hepatectomy with curative intention.

EphB3 is overexpressed in non-small-cell lung cancer and promotes tumor metastasis by enhancing cell survival and migration.

Eph receptors, the largest subfamily of transmembrane tyrosine kinase receptors, have been increasingly implicated in various physiologic and pathologic processes, and the roles of the Eph family members during tumorigenesis have recently attracted growing attention. Until now, research on EphB3 function in cancer is limited to focusing on tumor suppression by EphB receptors in colorectal cancer. However, its function in other types of cancer remains poorly investigated. In this study, we explored the function of EphB3 in non-small-cell lung cancer (NSCLC). We found that the expression of EphB3 was significantly upregulated in clinical samples and cell lines, and the expression level correlated with the patient pathologic characteristics, including tumor size, differentiation, and metastasis. Overexpression of EphB3 in NSCLC cell lines accelerated cell growth and migration and promoted tumorigenicity in xenografts in a kinase-independent manner. In contrast, downregulation of EphB3 inhibited cell proliferation and migration and suppressed in vivo tumor growth and metastasis. Furthermore, we showed that silencing of EphB3 inhibited cell growth by reducing DNA synthesis and caspase-8-mediated apoptosis and suppressed cell migration by increasing accumulation of focal adhesion formation. Taken together, our findings suggest that EphB3 provides critical support to the development and progression of NSCLC by stimulating cell growth, migration, and survival, thereby implicating EphB3 as a potential therapeutic target in NSCLC.

Connective tissue growth factor is overexpressed in esophageal squamous cell carcinoma and promotes tumorigenicity through beta-catenin-T-cell factor/Lef signaling.

Connective tissue growth factor (CTGF or CCN2), a member of the CCN family, is involved in diverse biological processes such as cell adhesion, proliferation, and angiogenesis. In this study, we show that overexpression of CTGF occurred in a significant proportion of esophageal squamous cell carcinoma (ESCC) samples that were of a high tumor grade and metastatic. Forced expression of CTGF in Eca109 ESCC cells accelerated their growth in culture and significantly increased tumor formation in nude mice, whereas RNA interference-mediated knockdown of CTGF in ESCC cells significantly inhibited cell growth and colony formation, as well as tumorigenicity in vivo. Moreover, overexpression of CTGF in ESCC cells resulted in the accumulation and nuclear translocation of beta-catenin, leading to activation of beta-catenin-T-cell factor (TCF)/Lef signaling. Up-regulation of c-Myc and cyclin D1, two target genes of beta-catenin-TCF/Lef signaling, was also observed in the CTGF-overexpressing cells. These effects of CTGF in ESCC cells were abolished by transfection with either dominant negative beta-catenin or dominant negative TCF4. Furthermore, we identified a beta-catenin-TCF/Lef-binding site (TBE) in the promoter region of CTGF and found that CTGF is a transcriptional target of beta-catenin-TCF/Lef signaling. Taken together, these results revealed that the interaction of CTGF and beta-catenin-TCF/Lef forms a positive feedback loop, which could contribute to the tumorigenicity of ESCC.