异常激活的葡萄糖-6-磷酸脱氢酶通过上调周期蛋白D1表达促进肾透明细胞癌增殖

葡萄糖6-磷酸脱氢酶（glucose 6-phosphate dehydrogenase,G6PD）为磷酸戊糖途径的调节酶。研究表明,G6PD与多种恶性肿瘤的发生密切相关。然而,G6PD在肾透明细胞癌（clear cell renal cell carcinoma, ccRCC）中的功能及其作用机制却鲜有报道。本研究通过TCGA数据分析发现,G6PD在肾透明细胞癌TNM Ⅲ/Ⅳ期mRNA表达水平显著升高,与患者的性别、原发肿瘤直径、淋巴结转移、远端转移、病灶一侧的偏重性、病理分级以及TNM临床分期密切相关。并且,G6PD异常激活有可能成为评价肾透明细胞癌患者不良预后的分子。细胞系检测结果提示,与对照293T细胞及恶性程度较低的786-O细胞相比,恶性程度较高的Caki-1细胞中的G6PD表达及活性明显增加。基因稳定转染结合CCK8分析结果显示,G6PD过表达或异常激活可显著提高293T及786-O细胞的增殖能力,并且促进786-O细胞中周期蛋白D1基因表达上调。综上,本研究通过TCGA数据库分析和稳定细胞系检测及CCK8分析,结果显示,G6PD在肾透明细胞癌中异常激活,并可上调细胞周期蛋白D1表达,进而促进肿瘤细胞增殖。该研究为进一步揭示肾透明细胞癌分子发病机制以及开发有效的靶向治疗方案提供了借鉴。     

Lipocalin2 Promotes Invasion,Tumorigenicity and Gemcitabine Resistance in Pancreatic Ductal Adenocarcinoma

Lipocalin 2 (LCN2) is a small secreted protein and its elevated expression has been observed in pancreatic as well as other cancer types. LCN2 has been reported to promote resistance to drug-induced apoptosis, enhance invasion through its physical association with matrix metalloproteinase-9, and promote in vivo tumor growth. LCN2 was found to be commonly expressed in patient PDAC samples and its pattern of immunohistochemical staining intensified with increasing severity in high-grade precursor lesions. Downregulation of LCN2 in two pancreatic ductal adenocarcinoma cell lines (BxPC3 and HPAF-II) with high LCN2 expression significantly reduced attachment, invasion, and tumour growth in vivo, but not proliferation or motility. Downregulation of LCN2 in two pancreatic ductal adenocarcinoma cell lines (BxPC3 and HPAF-II) with high expression significantly reduced attachment, invasion, and tumour growth in vivo. In contrast, LCN2 overexpression in PANC1, with low endogenous expression, significantly increased invasion, attachment, and enhanced tumor growth. Suppression of LCN2 in BxPC3 and HPAF-II cells increased their sensitivity to gemcitabine in vitro, and in vivo when BxPC3 was tested. Furthermore, LCN2 promotes expression of VEGF and HIF1A which contribute to enhanced vascularity. These overall results demonstrate that LCN2 plays an important role in the malignant progression of pancreatic ductal carcinoma and is a potential therapeutic target for this disease.     

ALKBH5-m6A-FOXM1 signaling axis promotes proliferation and invasion of lung adenocarcinoma cells under intermittent hypoxia

Obstructive sleep apnea (OSA) is closely associated with cancer progression and cancer-related mortality. N6-methyladenosine (m⁶A) is involved in the process of intermittent hypoxia (IH) promoting tumor progression. However, it is unclear how m⁶A regulates the development of lung adenocarcinoma under IH. In this study, we found that ALKBH5 was elevated in lung adenocarcinoma cells and subcutaneous tumors in mice under IH, which was associated with decreased m⁶A levels in these cells and tissues. Next, we knocked out ALKBH5 in a human lung adenocarcinoma cell line under IH, and we found that the proliferation and invasion of these cells were significantly inhibited. Mechanistic analysis showed that under IH, knockout of ALKBH5 in lung adenocarcinoma cells upregulated the level of m⁶A in Forkhead box M1 (FOXM1) mRNA and decreased the translation efficiency of FOXM1 mRNA, resulting in downregulation of the FOXM1 protein. The FOXM1 protein is elevated in lung adenocarcinoma cells and subcutaneous tumor tissues of mice under IH. By knocking out FOXM1 in lung adenocarcinoma cells under IH, proliferation and invasion of these cells were inhibited, and overexpression of FOXM1 partially restored the inhibition of growth and invasion of lung adenocarcinoma cells due to ALKBH5 knockout. Collectively, our findings demonstrate that the m⁶A demethylase ALKBH5 affects the proliferation and invasion of lung adenocarcinoma cells under IH by downregulating m⁶A modification on FOXM1 mRNA and by promoting FOXM1 expression.     

The Motor Protein KIF14 Inhibits Tumor Growth and Cancer Metastasis in Lung Adenocarcinoma

The motor protein kinesin superfamily proteins (KIFs) are involved in cancer progression. The depletion of one of the KIFs, KIF14, might delay the metaphase-to-anaphase transition, resulting in a binucleated status, which enhances tumor progression; however, the exact correlation between KIF14 and cancer progression remains ambiguous. In this study, using loss of heterozygosity and array comparative genomic hybridization analyses, we observed a 30% loss in the regions surrounding KIF14 on chromosome 1q in lung adenocarcinomas. In addition, the protein expression levels of KIF14 in 122 lung adenocarcinomas also indicated that approximately 30% of adenocarcinomas showed KIF14 down-regulation compared with the expression in the bronchial epithelial cells of adjacent normal counterparts. In addition, the reduced expression of KIF14 mRNA or proteins was correlated with poor overall survival (P = 0.0158 and <0.0001, respectively), and the protein levels were also inversely correlated with metastasis (P<0.0001). The overexpression of KIF14 in lung adenocarcinoma cells inhibited anchorage-independent growth in vitro and xenograft tumor growth in vivo. The overexpression and silencing of KIF14 also inhibited or enhanced cancer cell migration, invasion and adhesion to the extracellular matrix proteins laminin and collagen IV. Furthermore, we detected the adhesion molecules cadherin 11 (CDH11) and melanoma cell adhesion molecule (MCAM) as cargo on KIF14. The overexpression and silencing of KIF14 enhanced or reduced the recruitment of CDH11 in the membrane fraction, suggesting that KIF14 might act through recruiting adhesion molecules to the cell membrane and modulating cell adhesive, migratory and invasive properties. Thus, KIF14 might inhibit tumor growth and cancer metastasis in lung adenocarcinomas.     

miR-200 Inhibits lung adenocarcinoma cell invasion and metastasis by targeting Flt1/VEGFR1

The microRNA-200 (miR-200) family is part of a gene expression signature that predicts poor prognosis in lung cancer patients. In a mouse model of K-ras/p53-mutant lung adenocarcinoma, miR-200 levels are suppressed in metastasis-prone tumor cells, and forced miR-200 expression inhibits tumor growth and metastasis, but the miR-200 target genes that drive lung tumorigenesis have not been fully elucidated. Here, we scanned the genome for putative miR-200 binding sites and found them in the 3'-untranslated region (3'-UTR) of 35 genes that are amplified in human cancer. Mining of a database of resected human lung adenocarcinomas revealed that the levels of one of these genes, Flt1/VEGFR1, correlate inversely with duration of survival. Forced miR-200 expression suppressed Flt1 levels in metastasis-prone lung adenocarcinoma cells derived from K-ras/p53-mutant mice, and negatively regulated the Flt1 3'-UTR in reporter assays. Cancer-associated fibroblasts (CAFs) isolated from murine lung adenocarcinomas secreted abundant VEGF and enhanced tumor cell invasion in coculture studies. CAF-induced tumor cell invasion was abrogated by VEGF neutralization or Flt1 knockdown in tumor cells. Flt1 knockdown decreased the growth and metastasis of tumor cells in syngeneic mice. We conclude that miR-200 suppresses lung tumorigenesis by targeting Flt1.     

Overexpression of DTL enhances cell motility and promotes tumor metastasis in cervical adenocarcinoma by inducing RAC1-JNK-FOXO1 axis

Cervical adenocarcinoma is an important disease that affects young women and it has a high mortality and poor prognosis. Denticleless E3 ubiquitin protein ligase homolog (DTL) gene with oncogenic function has been evaluated in several cancers. Through this study, we aimed to clarify the clinical and molecular characteristics of cervical adenocarcinoma involving overexpression of DTL and elucidate its molecular mechanism. Bioinformatics analysis was performed through multiple databases. RNA sequencing was used to obtain differentially expressed genes after DTL was overexpressed in cells. The role of DTL in cervical adenocarcinoma was explored through in vitro and in vivo experiments. We found that DTL has an unfavorable prognostic implication for patients with cervical adenocarcinoma. Overexpression of DTL induced the migration and invasion of tumor cells in vitro and promoted intra-pulmonary metastasis in vivo. In addition, DTL activated JNK through RAC1 and upregulated FOXO1 to induce epithelial–mesenchymal transition, and the migration and invasion of tumor cells. Therefore, we conclude that overexpression of DTL enhanced cell motility and promoted tumor metastasis of cervical adenocarcinoma by regulating the RAC1-JNK-FOXO1 axis. These results suggest that DTL may become a potential therapeutic target for antitumor metastasis of cervical adenocarcinoma.Subject terms: Cancer epigenetics, Cervical cancer     

Overexpression of a novel regulator of p120 catenin,NLBP, promotes lung adenocarcinoma proliferation

NLBP (novel LZAP-binding protein) was recently shown to function as a tumor suppressor capable of inhibiting the NFκB signaling pathway. NLBP is also known as a negative regulator of cell invasion, and its expression is reduced in several cancer cell lines that have little invasive activity. Although these phenomena suggest that NLBP may be a potential tumor suppressor, its role as a tumor suppressor in human lung cancer is not well established. In contrast to our expectation, NLBP was highly expressed in the early stage of lung adenocarcinoma tissues, and overexpression of NLBP promoted proliferation of H1299 lung adenocarcinoma cells. We also found that p120 catenin (p120ctn) was a novel binding partner of NLBP, and that NLBP binds to the regulatory domain of p120ctn, and p120ctn associates with N-terminal region of NLBP, respectively. This binding leads to p120ctn stability to inhibit proteasomal degradation of p120ctn by inhibiting its ubiqutination. In addition, we also found that overexpression of NLBP and p120ctn in human lung cancer are closely related with adenocarcinoma compared with squamous cell carcinoma. Taken together, our findings reveal that NLBP is highly overexpressed in human lung adenocarcinoma, and that overexpression of NLBP promotes the cell proliferation of lung adenocarcinoma through interacting with p120ctn and suggest that NLBP may function as an oncogene in early stage carcinogenesis of lung adenocarcinoma.     

LncRNA TTN-AS1 drives invasion and migration of lung adenocarcinoma cells via modulation of miR-4677-3p/ZEB1 axis

Lung adenocarcinoma is the most prevalent type of lung cancer with a high incidence and mortality worldwide. Metastasis is the major cause of high death rate in lung cancer and the potential mechanism of lung adenocarcinoma metastasis remains indistinct. Emerging investigations have demonstrated that long noncoding RNA is a kind of non–protein coding RNA and plays a critical role in cancer progression and metastasis. TTN antisense RNA 1 (TTN-AS1) has been reported to promote cell growth and metastasis in cancer. However, the function of TTN-AS1 in lung adenocarcinoma is still to be illustrated. In this study, we observed that TTN-AS1 was upregulated in tissues and cells of lung adenocarcinoma and associated with poor overall survival. TTN-AS1 promoted cell proliferation, migration, invasion, and epithelial-mesenchymal transition in lung cancer. TTN-AS1 directly bound with miR-4677-3p and negatively regulated miR-4677-3p. MiR-4677-3p rescued the inhibitive impacts of TTN-AS1 knockdown on lung adenocarcinoma. Furthermore, zinc finger E-box binding homeobox 1 (ZEB1) was the target of miR-4677-3p, and TTN-AS1 modulated ZEB1 by competing for miR-4677-3p. TTN-AS1 drove the invasion and migration of lung adenocarcinoma cells by targeting the miR-4677-3p/ZEB1 axis. To sum up, our study offers insights into the mechanism of TTN-AS1 in lung adenocarcinoma metastasis and targeting the TTN-AS1/miR-4677-3p/ZEB1 axis may be the potential innovate therapeutic strategy for the patients with lung adenocarcinoma.     

FoxM1 Is Associated with Poor Prognosis of Non-Small Cell Lung Cancer Patients through Promoting Tumor Metastasis

Background

FoxM1 has been reported to be important in initiation and progression of various tumors. However, whether FoxM1 has any indication for prognosis in non-small cell lung cancer patients remains unclear.

Methodology/Principal Findings

In this study, FoxM1 expression in tumor cells was examined first by immunohistochemistry in 175 NSCLC specimens, the result of which showed that FoxM1 overexpression was significantly associated with positive smoking status (P = 0.001), poorer tissue differentiation (P = 0.0052), higher TNM stage (P<0.0001), lymph node metastasis (P<0.0001), advanced tumor stage (P<0.0001), and poorer prognosis (P<0.0001). Multivariable analysis showed that FoxM1 expression increased the hazard of death (hazard ratio, 1.899; 95% CI, 1.016–3.551). Furthermore, by various in vitro and in vivo experiments, we showed that targeted knockdown of FoxM1 expression could inhibit the migratory and invasive abilities of NSCLC cells, whereas enforced expression of FoxM1 could increased the invasion and migration of NSCLC cells. Finally, we found that one of the cellular mechanisms by which FoxM1 promotes tumor metastasis is through inducing epithelial-mesenchymal transition (EMT) program.

Conclusions

These results suggested that FoxM1 overexpression in tumor tissues is significantly associated with the poor prognosis of NSCLC patients through promoting tumor metastasis.     

MicroRNA-29a suppresses the growth,migration, and invasion of lung adenocarcinoma cells by targeting carcinoembryonic antigen-related cell adhesion molecule 6

Carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) is an important regulator of cell adhesion, invasion, and metastasis. The aim of this study was to evaluate the functional roles of CEACAM6 in lung adenocarcinoma and to identify miRNAs that inhibit the growth, migration, and invasion of lung adenocarcinoma cells by targeting CEACAM6. CEACAM6 expression is associated with poor prognosis of patients with lung adenocarcinoma, and CEACAM6 has important functional roles in controlling the growth, migration, and invasion of lung adenocarcinoma cells in vitro and in vivo. Furthermore, miR-29a can suppress the growth, migration, and invasion of lung adenocarcinoma cells by targeting CEACAM6. Therefore, miR-29a/CEACAM6 axis represents a potential therapeutic target for treatment of lung adenocarcinoma.     

MMP-2 VEGF在肺腺癌细胞中的表达及其意义

目的研究探讨基质金属蛋白酶2(MMP-2)及血管内皮生长因子(VEGF)在胸腔积液、痰液中肺腺癌细胞的不同表达及二者在肺癌细胞侵袭转移过程中的相互关系。方法选择胸腔积液、痰液共计264例癌性及异型增生细胞标本经免疫细胞化学方法分别检测MMP-2 VEGF的表达情况。结果免疫细胞化学结果显示:MMP-2在胸腔积液中腺癌细胞、异型增生上皮细胞的表达率分别为71.7%(99/138)、16.7%(6/36),在胸膜炎和结核病变典型良性胸腔积液增生上皮细胞中不表达;在痰腺癌细胞中的表达率为39.1%(27/69),统计结果显示MMP-2在恶性胸腔积液腺癌细胞中的表达率明显高于在异型增生的上皮、增生的上皮及痰腺癌细胞的表达率(P均0.05)。VEGF在胸腔积液中腺癌细胞、异型增生上皮细胞的表达率分别为89.1%(123/138)、33.3%(12/36),在胸膜炎和结核病变典型良性胸腔积液增生上皮细胞中不表达;在痰腺癌细胞中的表达率为47.8%(33/69),VEGF在恶性胸腔积液腺癌细胞中表达率明显高于在异型增生的上皮细胞、增生的上皮细胞及痰腺癌细胞的表达率(P均0.05),且MMP-2同VEGF总阳性表达率之间成正相关(r=0.867,P=0.049)。结果 MMP-2 VEGF在胸水腺癌细胞中高表达,可能与肺腺癌的转移、侵袭有关;两者联合做免疫细胞化学检查对肺腺癌细胞病理诊断有辅助意义。     

LncRNA LINC01512 Promotes the Progression and Enhances Oncogenic Ability of Lung Adenocarcinoma

Previously, a significantly upregulated lncRNA, LINC01512, in lung adenocarcinoma (LAD) was obtained, while its biological function and molecular mechanisms were unclear. The expression level of LINC01512 was estimated by qPCR from 100 pairs of LAD and NT samples. The correlation of LINC01512 to clinical data of LAD patients was analyzed. LINC01512 was knocked down and overexpressed in SPCA‐1 and A549 cell lines by lentivirus‐mediated technology, and the oncological behavioral changes of SPCA‐1 and A549 cells were observed, as well as, tumorigenicity in experimental nude mice. Compared to the adjacent tissues, LINC01512 was obviously upregulated in LAD. The expression level of LINC01512 was closely related to lymph node metastasis and tumor node metastasis (TNM) stage. Survival analysis showed that the survival time of high expression LINC01512 group was significantly shorter than the low‐expression group in LAD. Knockdown or overexpression test unanimously confirmed that LINC01512 can increase the ability of cell migration, invasion, proliferation, colony formation, adhesion, and S phase and G2/M phase cells, whereas decrease the apoptosis and G0/G1 phase cells. Nude mice experiments confirmed that LINC01512 significantly enhanced the speed and weight of tumorigenicity. LINC01512 is an oncogenic lncRNA gene that promotes the progression and distinctly enhances the oncogenic ability in lung adenocarcinoma. J. Cell. Biochem. 118: 3102–3110, 2017. © 2017 The Authors. Journal of Cellular Biochemistry Published by Wiley Periodicals Inc.     

丝甘蛋白聚糖促进非小细胞肺癌的侵袭与转移

丝甘蛋白聚糖(serglycin,SRGN)在肿瘤细胞的侵袭与转移中具有广泛的研究前景。本研究报道SRGN与肺癌细胞侵袭与转移能力之间的相关性。首先,通过检测SRGN在正常肺上皮细胞株BEAS-2B及不同侵袭与转移能力的肺癌细胞株95C、95D中的表达差异。利用shRNA干扰技术,在侵袭与转移能力强的95D细胞中建立稳定干扰SRGN表达的95D/shSRGN的细胞株,并通过RT-qPCR、Western印迹、酶联免疫吸附测定验证其敲除效率。结果显示：干扰SRGN可抑制侵袭与转移性强的95D细胞的侵袭与转移能力,减弱细胞迁移与侵袭等生物学特性,导致上皮标志物上皮细胞钙黏连蛋白（E-cadherin）表达上调,间质标志物纤维连接蛋白1（fibronectin1, FN1）及EMT（epithelial-mesenchymal transition, EMT）相关转录因子锌指E盒结合同源框1（zinc finger E-box binding homeobox 1, ZEB1）表达下调。进一步分析发现, SRGN与上皮细胞钙黏连蛋白表达成负相关（P=-0.25）,而与FN1(P=0.12)及ZEB1(P=0.35)表达成正相关,并且SRGN高表达的患者总生存时间明显少于SRGN低表达组（P=0.0077）,SRGN与ZEB1同时高表达的患者,总生存时间显著小于SRGN与ZEB1低表达患者（P=0.0005）。研究结果证实,SRGN促进上皮间质转化发生,增强非小细胞肺癌的侵袭与转移能力,为非小细胞肺癌预后提供参考。     

MiR-186-5p通过靶向调控PTTG1抑制肺腺癌细胞的上皮-间质转化

先前研究表明,miR-186-5p在人类许多恶性肿瘤中扮演抑癌基因的作用,但其在肺腺癌上皮-间质转化（epithelial-mesenchymal transition,EMT）中的作用并不明确。本研究旨在证明,miR-186-5p可通过靶向调控PTTG1抑制肺腺癌细胞的上皮-间质转化。我们首先分析了miR-186-5p在人肺癌细胞中的表达。荧光定量PCR（QRT-PCR）结果显示,与人正常肺上皮细胞BEAS-2B相比,肺腺癌细胞SPC-A1、A549中的miR-186-5p表达量明显降低。为研究miR-186-5p在肺腺癌细胞中的功能,利用GV369-miR-186-5p表达载体,实现了在A549细胞中的过表达。基因转染结合Transwell侵袭结果显示,与对照质粒转染的A549细胞相比,过表达miR-186-5p的A549细胞的体外侵袭能力明显下降。Western印迹检测细胞中EMT相关标志物揭示,GV369-miR-186-5p转染的A549细胞中的上皮-钙黏着蛋白（E-cadherin）表达明显上调,而神经-钙黏着蛋白（N-cadherin）和波形蛋白（vimentin）表达明显下调。同时,GV369-miR-186-5p转染引起其靶基因--垂体肿瘤转化基因1（pituitary tumor-transforming gene 1,PTTG1）编码蛋白在A549细胞中明显降低。重要的是,过表达miR-186-5p与敲减PTTG1均可导致上皮-钙黏着蛋白表达上调,而神经-钙黏着蛋白和波形蛋白下调|而miR-186-5p和PTTG1表达载体共转染后,3种EMT相关标志物在A549的表达与对照细胞的表达无明显差异,提示过表达PTTG1可抵消miR-186-5p对EMT相关标志物表达的影响。综上所述,miR-186-5p可通过靶向调控PTTG1抑制EMT的发生,进而抑制肺腺癌细胞的侵袭转移。     

Prognostic Importance and Therapeutic Implications of PAK1, a Drugable Protein Kinase,in Gastroesophageal Junction Adenocarcinoma

Gastroesophageal junction (GEJ) adenocarcinoma is a lethal cancer with rising incidence, yet the molecular biomarkers that have strong prognostic impact and also hold great therapeutic promise remain elusive. We used a data mining approach and identified the p21 protein-activated kinase 1 (PAK1), an oncogene and drugable protein kinase, to be among the most promising targets for GEJ adenocarcinoma. Immunoblot analysis and data mining demonstrated that PAK1 protein and mRNA were upregulated in cancer tissues compared to the noncancerous tissues. Immunohistochemistry revealed PAK1 overexpression in 72.6% of primary GEJ adenocarcinomas (n = 113). A step-wise increase in PAK1 levels was noted from paired normal epithelium, to atypical hyperplasia and adenocarcinoma. PAK1 overexpression in tumor was associated with lymph node (LN) metastasis (P<0.001), advanced tumor stage (P<0.001), large tumor size (P = 0.006), residual surgical margin (P = 0.033), and unfavorable overall survival (P<0.001). Multivariate analysis showed PAK1 overexpression is an independent high-risk prognostic predictor (P<0.001). Collectively, PAK1 is overexpressed during tumorigenic progression and its upregulation correlates with malignant properties mainly relevant to invasion and metastasis. PAK1 expression could serve as a prognostic predictor that holds therapeutic promise for GEJ adenocarcinoma.