ROR1激活AKT/FOXO1信号介导非小细胞肺癌吉非替尼耐药

EGFR-TKI靶向治疗在非小细胞肺癌（non-small cell lung cancer, NSCLC）综合治疗中显示出重要作用;然而,耐药性却极大限制其临床治疗效果。受体酪氨酸激酶样孤儿受体（receptor tyrosine kinase-like orphan receptor 1, ROR1）是I型受体酪氨酸激酶家族中的成员,在肿瘤发生发展中发挥重要作用。本研究拟探讨ROR1介导非小细胞肺癌吉非替尼耐药的作用及机制。采用吉非替尼反复诱导非小细胞肺癌HCC827细胞,建立吉非替尼耐药细胞株HCC827/GR。应用荧光定量PCR和Western 印迹检测HCC827/GR内ROR1的表达。采用shRNA的方法体外检测ROR1敲除前后HCC827/GR对吉非替尼耐药的变化,采用体外检测ROR1过表达前后HCC827对吉非替尼耐药的变化。体内检测ROR1敲除前后HCC827/GR对吉非替尼耐药的变化。Western 印迹检测HCC827/GR内ROR1下游信号分子的活化。实时荧光定量PCR及Western 印迹结果显示,HCC827/GR耐药细胞中的ROR1 mRNA和蛋白质表达水平显著高于HCC827敏感细胞。体外干扰ROR1表达,可明显增强HCC827/GR耐药细胞对吉非替尼的敏感性 (IC50 15.3±3.69 vs. 4.2±1.38),增加吉非替尼诱导的细胞凋亡 (20.5±2.52 vs. 41.8±3.74)。体外过表达ROR1显著增强HCC827敏感细胞对吉非替尼的耐药性(IC50 0.8±0.52 vs. 2.2±0.87)。体内裸鼠移植瘤实验同样发现,干扰ROR1能增强HCC827/GR移植瘤对吉非替尼的敏感性。进一步研究发现,AKT/FOXO1信号在HCC827/GR耐药细胞中异常活化,而干扰ROR1能够抑制AKT的磷酸化,并上调FOXO1的表达。上述结果表明,ROR1参与非小细胞肺癌吉非替尼耐药,抑制ROR1能够逆转吉非替尼耐药,其机制与ROR1调控AKT/FOXO1信号有关。     

Promoter methylation of argininosuccinate synthetase-1 sensitises lymphomas to arginine deiminase treatment, autophagy and caspase-dependent apoptosis

Tumours lacking argininosuccinate synthetase-1 (ASS1) are auxotrophic for arginine and sensitive to amino-acid deprivation. Here, we investigated the role of ASS1 as a biomarker of response to the arginine-lowering agent, pegylated arginine deiminase (ADI-PEG20), in lymphoid malignancies. Although ASS1 protein was largely undetectable in normal and malignant lymphoid tissues, frequent hypermethylation of the ASS1 promoter was observed specifically in the latter. A good correlation was observed between ASS1 methylation, low ASS1 mRNA, absence of ASS1 protein expression and sensitivity to ADI-PEG20 in malignant lymphoid cell lines. We confirmed that the demethylating agent 5-Aza-dC reactivated ASS1 expression and rescued lymphoma cell lines from ADI-PEG20 cytotoxicity. ASS1-methylated cell lines exhibited autophagy and caspase-dependent apoptosis following treatment with ADI-PEG20. In addition, the autophagy inhibitor chloroquine triggered an accumulation of light chain 3-II protein and potentiated the apoptotic effect of ADI-PEG20 in malignant lymphoid cells and patient-derived tumour cells. Finally, a patient with an ASS1-methylated cutaneous T-cell lymphoma responded to compassionate-use ADI-PEG20. In summary, ASS1 promoter methylation contributes to arginine auxotrophy and represents a novel biomarker for evaluating the efficacy of arginine deprivation in patients with lymphoma.     

The TTYH3/MK5 Positive Feedback Loop regulates Tumor Progression via GSK3-β/β-catenin signaling in HCC

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related death worldwide, and identification of novel targets is necessary for its diagnosis and treatment. This study aimed to investigate the biological function and clinical significance of tweety homolog 3 (TTYH3) in HCC. TTYH3 overexpression promoted cell proliferation, migration, and invasion and inhibited HCCM3 and Hep3B cell apoptosis. TTYH3 promoted tumor formation and metastasis in vivo. TTYH3 upregulated calcium influx and intracellular chloride concentration, thereby promoting cellular migration and regulating epithelial-mesenchymal transition-related protein expression. The interaction between TTYH3 and MK5 was identified through co-immunoprecipitation assays and protein docking. TTYH3 promoted the expression of MK5, which then activated the GSK3β/β-catenin signaling pathway. MK5 knockdown attenuated the activation of GSK3β/β-catenin signaling by TTYH3. TTYH3 expression was regulated in a positive feedback manner. In clinical HCC samples, TTYH3 was upregulated in the HCC tissues compared to nontumor tissues. Furthermore, high TTYH3 expression was significantly correlated with poor patient survival. The CpG islands were hypomethylated in the promoter region of TTYH3 in HCC tissues. In conclusion, we identified TTYH3 regulates tumor development and progression via MK5/GSK3-β/β-catenin signaling in HCC and promotes itself expression in a positive feedback loop.     

Epithelial argininosuccinate synthetase is dispensable for intestinal regeneration and tumorigenesis

The epithelial signaling pathways involved in damage and regeneration, and neoplastic transformation are known to be similar. We noted upregulation of argininosuccinate synthetase (ASS1) in hyperproliferative intestinal epithelium. Since ASS1 leads to de novo synthesis of arginine, an important amino acid for the growth of intestinal epithelial cells, its upregulation can contribute to epithelial proliferation necessary to be sustained during oncogenic transformation and regeneration. Here we investigated the function of ASS1 in the gut epithelium during tissue regeneration and tumorigenesis, using intestinal epithelial conditional Ass1 knockout mice and organoids, and tissue specimens from colorectal cancer patients. We demonstrate that ASS1 is strongly expressed in the regenerating and Apc-mutated intestinal epithelium. Furthermore, we observe an arrest in amino acid flux of the urea cycle, which leads to an accumulation of intracellular arginine. However, loss of epithelial Ass1 does not lead to a reduction in proliferation or increase in apoptosis in vivo, also in mice fed an arginine-free diet. Epithelial loss of Ass1 seems to be compensated by altered arginine metabolism in other cell types and the liver.Subject terms: Cancer metabolism, Regeneration, Cancer metabolism, Experimental models of disease, Oncogenesis     

MicroRNA-1271 functions as a potential tumor suppressor in hepatitis B virus–associated hepatocellular carcinoma through the AMPK signaling pathway by binding to CCNA1

Hepatocellular carcinoma (HCC) is mainly associated with hepatitis B virus (HBV) infection and characterized by metastasizing and infiltrating adjacent and distant tissues. Notably, microRNA-1271 (miR-1271) is a tumor suppressor in various cancers. Therefore, we evaluate the ability of miR-1271 to influence cell proliferation, migration, invasion, and apoptosis in HBV-associated HCC through the Adenosine monophosphate–activated protein kinase (AMPK) signaling pathway via targeting CCNA1. HBV-associated HCC and adjacent normal tissues were collected to identify the expression of miR-1271 and CCNA1. To verify the relationship between miR-1271 and CCNA1, we used bioinformatics prediction and the dual-luciferase reporter gene assay. The effects of miR-1271 on HBV-associated HCC cell behaviors were investigated by treatment of the miR-1271 mimic, the miR-1271 inhibitor, or small interfering RNA against CCNA1. The HBV-DNA quantitative assay, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromid assay, scratch test, transwell assay, and flow cytometry were used to detect HBV-DNA replication, cell proliferation, invasion, migration, and apoptosis. MiR-1271 showed a low expression, whereas CCNA1 showed a high expression in HBV-associated HCC tissues. We identified that miR-1271 targeted and negatively regulated CCNA1. Upregulated miR-1271 and downregulated CCNA1 inhibited the HBV-associated HCC cell HBV-DNA replication, proliferation, migration, and invasion, while accelerating apoptosis by activating the AMPK signaling pathway. MiR-1271 promotes the activation of the AMPK signaling pathway by binding to CCNA1, whereby miR-1271 suppresses HBV-associated HCC progression. This study points to a potential therapeutic approach of downregulation of miR-1271 in HBV-associated HCC treatment.     

SLPI suppresses hepatocellular carcinoma progression via endoplasmic reticulum stress induced apoptosis

Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide. Secretory leukocyte protease inhibitor (SLPI) has been reported to function as a regulatory factor in several cancers. However, its biological functions and underlying mechanisms in HCC remain to be uncovered. Here, we aimed to explore the effect of SLPI in HCC. In our study, we found that the mRNA and protein expression levels of SLPI were significantly down-regulated in HCC tissues and hepatoma cell lines and low level of SLPI predicted worse survival in our HCC cohorts. In term of function, silencing of SLPI markedly promoted whereas overexpression SLPI suppressed proliferation, migration and invasion capabilities of HCC cells in vitro, and ectopic expression of SLPI inhibited the tumorigenicity of HCC cells in vivo. Mechanistic studies demonstrated that SLPI played a protective role in HCC progression via activating endoplasmic reticulum stress (ER stress)-mediated apoptosis of hepatoma cells, which could be regulated by MAPK signaling pathways. In summary, our findings highlight that SLPI could serve as a potential prognostic biomarker and putative tumor suppressor by enhancing ER stress-induced apoptosis in HCC cells mediated by MAPK signaling pathways, which provides new insights into promising therapeutic targets for HCC treatment.     

Deoxyribonuclease 1-like 3 Inhibits Hepatocellular Carcinoma Progression by Inducing Apoptosis and Reprogramming Glucose Metabolism

HCC has remained one of the challenging cancers to treat, owing to the paucity of drugs targeting the critical survival pathways. Considering the cancer cells are deficient in DNase activity, the increase of an autonomous apoptisis endonuclease should be a reasonable choice for cancer treatment. In this study, we investigated whether DNASE1L3, an endonuclease implicated in apoptosis, could inhibit the progress of HCC. We found DNASE1L3 was down-regulated in HCC tissues, whereas its high expression was positively associated with the favorable prognosis of patients with HCC. Besides, serum DNASE1L3 levels were lower in HCC patients than in healthy individuals. Functionally, we found that DNASE1L3 inhibited the proliferation of tumor cells by inducing G0/G1 cell cycle arrest and cell apoptosis in vitro. Additionally, DNASE1L3 overexpression suppressed tumor growth in vivo. Furthermore, we found that DNASE1L3 overexpression weakened glycolysis in HCC cells and tissues via inactivating the rate-limiting enzymes involved in PTPN2-HK2 and CEBPβ-p53-PFK1 pathways. Finally, we identified the HBx to inhibit DNASE1L3 expression by up-regulating the expression of ZNF384. Collectively, our findings demonstrated that DNASE1L3 could inhibit the HCC progression through inducing cell apoptosis and weakening glycolysis. We believe DNASE1L3 could be considered as a promising prognostic biomarker and therapeutic target for HCC.     

Reciprocal Activating Crosstalk between c-Met and Caveolin 1 Promotes Invasive Phenotype in Hepatocellular Carcinoma

c-Met, the receptor for Hepatocyte Growth Factor (HGF), overexpressed and deregulated in Hepatocellular Carcinoma (HCC). Caveolin 1 (CAV1), a plasma membrane protein that modulates signal transduction molecules, is also overexpressed in HCC. The aim of this study was to investigate biological and clinical significance of co-expression and activation of c-Met and CAV1 in HCC. We showed that c-Met and CAV1 were co-localized in HCC cells and HGF treatment increased this association. HGF-triggered c-Met activation caused a concurrent rise in both phosphorylation and expression of CAV1. Ectopic expression of CAV1 accelerated c-Met signaling, resulted in enhanced migration, invasion, and branching-morphogenesis. Silencing of CAV1 downregulated c-Met signaling, and decreased migratory/invasive capability of cells and attenuated branching morphogenesis. In addition, activation and co-localization of c-Met and CAV1 were elevated during hepatocarcinogenesis. In conclusion reciprocal activating crosstalk between c-Met and CAV1 promoted oncogenic signaling of c-Met contributed to the initiation and progression of HCC.     

Cks1 regulates human hepatocellular carcinoma cell progression through osteopontin expression

Precise cell cycle regulation is critical to prevent aberrant cell proliferation and cancer progression. Cks1 was reported to be an essential accessory factor for SCF^Skp2, the ubiquitin ligase that targets p27^Kip1 for proteasomal degradation; these actions drive mammalian cell transition from G1 to S phase. In this study, we investigated the role played by Cks1 in the growth and progression of human hepatocellular carcinoma (HCC) cells. Silencing Cks1 expression abrogated osteopontin (OPN) expression in a p27^Kip1-dependent manner in Huh7 HCC cells. OPN increased the proliferation, migration and invasion of Huh7 cells. Pharmacological inhibitor studies demonstrated that ERK1/2 signaling is responsible mainly for Cks1-mediated OPN expression. Cks1 appears to regulate ERK1/2 signaling through the expression of dual-specificity phosphatase 16 (DUSP16) because both Cks1 knockdown, which leads to DUSP16 upregulation, and DUSP16 overexpression decreased ERK1/2 phosphorylation and the resulting OPN expression. The same is true for the Cks1-mediated increases in p27^Kip1, suggesting that Cks1 regulates OPN expression through activating ERK1/2 signaling either by suppressing DUSP16 expression or by a p27^Kip1-dependent mechanism. Cks1 and OPN expression levels were significantly higher, but DUSP16 expression levels were significantly lower in HCC tissues than in normal liver tissues. Both Cks1 and OPN expression were negatively correlated with DUSP16 expression, whereas Cks1 expression was positively correlated with OPN expression. Moreover, combined panels for the expression levels of Cks1, DUSP16 and OPN showed significant prognostic power for the risk assessment of HCC patient overall survival. In conclusion, our data propose a novel function for Cks1 as a tumor promoter through the expression of the strongly oncogenic protein OPN in HCC.     

Modulation of formation of the 3’-end of the human argininosuccinate synthetase mRNA by GT-repeat polymorphism

Microsatellites are abundant in the human genome and may acquire context-dependent functions. A highly polymorphic GT microsatellite is located downstream of the poly(A) signal of the human argininosuccinate synthetase (ASS1) gene. The ASS1 participates in urea and nitric oxide production and is a rate-limiting enzyme in arginine biosynthesis. To examine possible involvement of the GT microsatellite in ASS1 mRNA 3’-end formation, ASS1 minigene constructs were used in transient transfection for assessment of poly(A) site usage by S1 nuclease mapping. Synthesis of the major human ASS1 mRNA is found to be controlled by two consecutive non-canonical poly(A) signals, UAUAAA and AUUAAA, located 7 nucleotides apart where a U-rich sequence and the GU microsatellite serve as their respective downstream GU/U-rich elements. Moreover, AUUAAA utilization is affected by the GU-repeat number possibly leading to differential regulation of ASS1 polyadenylation in individuals with different repeat numbers. Interestingly, the less efficient UAUAAA motif is noted to be the major ASS1 poly(A) signal possibly as a result of an indispensable downstream U-rich element and restricted utilization of the AUUAAA motif by the presence of extended GU-repeats. The UAUAAA motif and the GT microsatellite are conserved only in primates whereas AUUAAA motif is present in all mammals analyzed. The suboptimal UAUAAA motif and the utilization of the polymorphic GT microsatellite as polyadenylation signal of the ASS1 gene may be used as a strategy in primates to modulate ASS1 level in response to interactions of genetic and environmental factors.     

α-Mangostin induces mitochondrial dependent apoptosis in human hepatoma SK-Hep-1 cells through inhibition of p38 MAPK pathway

α-Mangostin is a dietary xanthone that has been shown to have anti-cancer and anti-proliferative properties in various types of human cancer cells. This study investigates the molecular mechanism of the apoptosis-inducing effects of α-mangostin on human hepatocellular carcinoma (HCC) cells. We observed that α-mangostin reduces the viability of HCC cells in a dose- and time-dependent manner. α-Mangostin mediated apoptosis of SK-Hep-1 cells is accompanied by nuclear chromatin condensation and cell cycle arrest in the sub-G1 phases as well as phosphatidylserine exposure. Furthermore, α-mangostin triggered the mitochondrial caspase apoptotic pathway, as indicated by the loss of mitochondrial membrane potential, the release of cytochrome c from mitochondria, and the regulation of B cell lymphoma 2 family member expression. Moreover, α-mangostin inhibited a sustained activation of p38 mitogen-activated protein kinase (MAPK) phosphorylation, and treatment with a p38 MAPK inhibitor enhanced α-mangostin-induced caspase activation and apoptosis in SK-Hep-1 cells. In vivo xenograft mice experiments revealed that α-mangostin significantly reduced tumor growth and weight in mice inoculated with SK-Hep-1 cells. These findings demonstrate that α-mangostin induces mitochondria-mediated apoptosis through inactivation of the p38 MAPK signaling pathway and that α-mangostin inhibits the in vivo tumor growth of SK-Hep-1 xenograft mice.     

MYO18B promotes hepatocellular carcinoma progression by activating PI3K/AKT/mTOR signaling pathway

Background

MYO18B has been identified as a novel tumor suppressor gene in several cancers. However, its specific roles in the progression of hepatocellular carcinoma (HCC) has not been well defined.

Methods

We firstly identified the expression and prognostic values of MYO18B in HCC using TCGA cohort and our clinical data. Then, MYO18B knockdown by RNA inference was implemented to investigate the effects of MYO18B on HCC cells. Quantitative RT-PCR and Western blot were used to determine gene and protein expression levels. CCK-8 and colony formation assays were performed to examine cell proliferation capacity. Wound healing and transwell assays were used to evaluate the migration and invasion of HepG2 cells.

Results

MYO18B was overexpressed and correlated with poor prognosis in HCC. MYO18B expression was an independent risk factor for overall survival. Knockdown of MYO18B significantly inhibited the proliferation, migration and invasion of HepG2 cells. Meanwhile, MYO18B knockdown could effectively suppress the phosphorylation of PI3K, AKT, mTOR and P70S6K, suggesting that MYO18B might promote HCC progression by targeting PI3K/AKT/mTOR signaling pathway.

Conclusions

MYO18B promoted tumor growth and migration via the activation of PI3K/AKT/mTOR signaling pathway. MYO18B might be a promising target for clinical intervention of HCC.

     

An RGD-Modified MRI-Visible Polymeric Vector for Targeted siRNA Delivery to Hepatocellular Carcinoma in Nude Mice

RNA interference (RNAi) has significant therapeutic promise for the genetic treatment of hepatocellular carcinoma (HCC). Targeted vectors are able to deliver small interfering RNA (siRNA) into HCC cells with high transfection efficiency and stability. The tripeptide arginine glycine aspartic acid (RGD)-modified non-viral vector, polyethylene glycol-grafted polyethylenimine functionalized with superparamagnetic iron oxide nanoparticles (RGD-PEG-g-PEI-SPION), was constructed as a magnetic resonance imaging (MRI)-visible nanocarrier for the delivery of Survivin siRNA targeting the human HCC cell line Bel-7402. The biophysical characterization of the RGD-PEG-g-PEI-SPION was performed. The RGD-modified complexes exhibited a higher transfection efficiency in transferring Survivin siRNA into Bel-7402 cells compared with a non-targeted delivery system, which resulted in more significant gene suppression at both the Survivin mRNA and protein expression levels. Then, the level of caspase-3 activation was significantly elevated, and a remarkable level of tumor cell apoptosis was induced. As a result, the tumor growth in the nude mice Bel-7402 hepatoma model was significantly inhibited. The targeting ability of the RGD-PEG-g-PEI-SPION was successfully imaged by MRI scans performed in vitro and in vivo. Our results strongly indicated that the RGD-PEG-g-PEI-SPION can potentially be used as a targeted non-viral vector for altering gene expression in the treatment of hepatocellular carcinoma and for detecting the tumor in vivo as an effective MRI probe.     

猕猴发育过程中肠肝组织血管活性肠肽及其受体的变化

本文旨在探讨猕猴发育过程中血管活性肠肽(vasoactive intestinal polypeptide,VIP)及其受体在肠肝组织的变化。通过手术途径获得胚胎6月、新生2 d、新生45 d和成年猕猴的回肠、肝脏、门静脉和外周血等标本,应用放射免疫分析法测定各标本中的VIP含量;通过免疫组化方法观察VIP在肠、肝组织内的分布;利用原位杂交法检测VIP受体1(VIP receptor 1,VIPR1)的表达。结果显示:(1)胚胎6月的猕猴小肠VIP含量为(20．7±14．3)ng／mg蛋白;小肠绒毛根部及黏膜下层可见少量的VIP阳性染色颗粒;在发育过程中,小肠VIP含量逐渐增加,成年期时达(514．8±49．2)ng／mg蛋白,较胚胎6月显著增加(P<0．01)。(2)成年猕猴小肠VIP主要分布于绒毛隐窝部、黏膜下层神经及环、纵行肌间神经丛及环行肌,在发育过程中相应部位的VIPR1表达逐渐上调。(3)肝脏在发育过程中VIP及VIPR1含量逐渐降低。(4)发育的各个时期,小肠组织的VIP含量均明显高于肝脏组织,门静脉VIP水平也始终高于外周血。结果提示,小肠绒毛隐窝部、黏膜下层神经及环、纵行肌间神经内VIP及VIPR1含量足在出生以后才迅速增加的;不论是在胚胎还是成年期,VIP均不在肝中代谢和分解,VIPR1仅见于胚胎肝脏血管。