结肠癌转移相关基因1与肿瘤

 结肠癌转移相关基因1（MACC1）为新近发现的调节肿瘤生长和转移的基因,在多种恶性肿瘤中异常表达,其编码蛋白作为HGF-MET信号通路的一个关键调节因子,可明显增强肿瘤细胞的侵袭及转移能力。     

肿瘤血管生成拟态在结肠癌中的研究进展

血管生成拟态（vasculogenic mimicry,VM）是近年来发现的一种与经典的肿瘤血管生成途径完全不同、不依赖机体内皮细胞的全新肿瘤微循环模式,与肿瘤生长、侵袭、转移及患者预后密切相关。VM可能是一个潜在的肿瘤治疗新靶点,但其形成的分子机制尚未完全清楚。本文就近年来VM在结肠癌中发现、可能分子机制及其对治疗的影响作一综述,探讨当前研究中存在的问题,展望未来的发展方向。     

Dickkopf-1 对结肠癌组织血管生成拟态形成的抑制作用及相关机制研究*

目的：探讨结肠癌组织中Dickkopf-1（Dkk 1）表达对血管生成拟态（vasculogenic mimicry ,VM）的抑制作用及相关机制。方法：收集2002年1 月至2004年12月间天津医科大学肿瘤医院收治的217 例结肠癌患者资料,利用CD34-PAS对结肠癌组织进行双重染色及免疫组化方法检测并分析VM与Dkk 1 表达的关系;利用体外三维培养（three dimensional culture,3Dculture）观察Dkk 1 对人结肠癌HCT 116 细胞管状结构形成能力及血管内皮钙黏蛋白（VE-cadherin）表达的影响;建立裸鼠皮下移植瘤模型进一步明确Dkk 1 对肿瘤组织内VM形成的抑制作用。结果：存在VM的结肠癌组织中Dkk 1 表达较低（P < 0.05）;Dkk 1 过表达的HCT 116 细胞形成管状结构能力明显减弱,且VE-cadherin表达降低;Dkk 1 可抑制裸鼠移植瘤组织中HCT 116 细胞形成VM。结论：Dkk 1 过表达可抑制结肠癌中VM形成。

     

Anti-angiogenic treatment promotes triple-negative breast cancer invasion via vasculogenic mimicry

Agents that target angiogenesis have shown limited efficacy for human triple-negative breast cancer (TNBC) in clinical trials. Along with endothelium-dependent vessels, there is also vasculogenic mimicry (VM) in the microcirculation of malignant tumors. The role of VM is not completely understood regarding anti-angiogenic treatment. In this study, human TNBC MDA-MB-231 and Hs578T and non-TNBC MCF-7 and BT474 tumor-bearing mice were treated with sunitinib, an anti-angiogenic drug, using a clinically relevant schedule. The drug was administered for one week and then discontinued. Tumor growth and invasion were observed, and the microcirculation patterns were detected with PAS/endomucin staining. Moreover, hypoxia and VM-associated proteins were evaluated with Hypoxyprobe kits and immunohistochemistry, respectively. Sunitinib significantly inhibited tumor growth in the TNBC and non-TNBC tumors. However, MDA-MB-231 and Hs578T tumors regrew and were more aggressive when the treatment was stopped. The discontinuation had no significant effect on the behavior of the non-TNBC MCF-7 and BT474 tumors. The growth of endothelium-dependent vessels in the TNBC MDA-MB-231 and Hs578T tumors were blocked by sunitinib, during which the number of VM channels significantly increased and resulted in a rebound of endothelium-dependent vessels after sunitinib discontinuation. Moreover, the VM-associated proteins VE-cadherin and Twist1 upregulated in the sunitinib-treated MDA-MB-231 and Hs578T tumors. Furthermore, the clinical significance of this upregulation was validated in 174 human breast cancers. The results from human breast cancer specimens indicated that there were more VM-positive TNBC cases than those in non-TNBC cases. HIF-1α, MMP2, VE-cadherin, and Twist1 were also expressed in a higher level in human TNBC compared with non-TNBC. In aconclusion, sunitinib promoted TNBC invasion by VM. The VM status could be helpful to predict the efficacy of anti-angiogenic therapy in patients with TNBC.     

CircMAN1A2 promotes vasculogenic mimicry of nasopharyngeal carcinoma cells through upregulating ERBB2 via sponging miR-940

Nasopharyngeal carcinoma (NPC) is the most prevalent human primary malignancy of the head and neck, and the presence of vasculogenic mimicry (VM) renders anti-angiogenic therapy ineffective and poorly prognostic. However, the underlying mechanisms are unclear. In the present study, we used miR-940 silencing and overexpression for in vitro NPC cell EdU staining, wound healing assay and 3D cell culture assay, and in vivo xenograft mouse model and VM formation to assess miR-940 function. We found that ectopic miR-940 expression reduced NPC cell proliferation, migration and VM, as well as tumorigenesis in vivo. By bioinformatic analysis, circMAN1A2 was identified as a circRNA that binds to miR-940. Mechanistically, we confirmed that circMAN1A2 acts as a sponge for miR-940, impairs the inhibitory effect of miR-940 on target ERBB2, and then activates the PI3K/AKT/mTOR signaling pathway using RNA-FISH, dual luciferase reporter gene and rescue analysis assays. In addition, upregulation of ERBB2 expression is associated with clinical staging and poor prognosis of NPC. Taken together, the present findings suggest that circMAN1A2 promotes VM formation and progression of NPC through miR-940/ERBB2 axis and further activates the PI3K/AKT/mTOR pathway. Therefore, circMAN1A2 may become a biomarker and therapeutic target for anti-angiogenic therapy in patients with nasopharyngeal carcinoma.     

Long noncoding RNA n339260 promotes vasculogenic mimicry and cancer stem cell development in hepatocellular carcinoma

Vasculogenic mimicry (VM) refers to the unique capability of aggressive tumor cells to mimic the pattern of embryonic vasculogenic networks. Cancer stem cells (CSC) represent a subpopulation of tumor cells endowed with the capacity for self‐renewal and multilineage differentiation. Previous studies have indicated that CSC may participate in the formation of VM. With the advance of high‐resolution microarrays and massively parallel sequencing technology, long noncoding RNAs (lncRNAs) are suggested to play a critical role in tumorigenesis and, in particular, the development of human hepatocellular carcinoma (HCC). Currently, no definitive relationship between lncRNA and VM formation has been described. In the current study, we demonstrated that expression of the lncRNA, n339260, is associated with CSC phenotype in HCC, and n339260 level correlated with VM, metastasis, and shorter survival time in an animal model. Overexpression of n339260 in HepG2 cells was associated with a significant increase in CSC. Additionally, the appearance of VM and vascular endothelial (VE)‐cadherin, a molecular marker of VM, was also induced by n339260 overexpression. Using a short hairpin RNA approach, n339260 was silenced in tumor cells, and knockdown of n339260 was associated with reduced VM and CSC. The results of this study indicate that n339260 promotes VM, possibly by the development of CSC. The related molecular pathways may be used as novel therapeutic targets for the inhibition of HCC angiogenesis and metastasis.     

结肠癌转移相关基因1及其在肿瘤中的作用

结肠癌转移相关基因1(MACC1)编码的蛋白质在肝细胞生长因子/肝细胞生长因子受体(HGF/c-Met)信号转导通路中转录激活c-Met基因,上调c-Met蛋白的表达,促进肿瘤细胞的侵袭和转移.MACC1蛋白高表达与人类多种肿瘤的发生和转移相关,如结肠癌、胃癌、肝癌、肺癌、卵巢癌等,且与肿瘤临床分期、有无远处转移密切相关,可作为肿瘤转移和预后判断的独立指标,为基因治疗提供新靶点.     

血管生成拟态与子宫颈癌的研究进展

血管生成拟态(VM)作为肿瘤中一种新的不依赖于血管内皮细胞的血液供应模式近年来备受关注.诸如缺氧、上皮间质转化(EMT)、肿瘤干细胞(CSCs)的存在等许多因素在VM的形成过程及肿瘤包括子宫颈癌的进展中发挥重要作用.研究VM及其影响因素在宫颈癌进展的作用机制为其复发、转移的治疗提供新思路.     

Claudin-4 is required for vasculogenic mimicry formation in human breast cancer cells

Vasculogenic mimicry (VM) refers to the unique capability of aggressive tumor cells to mimic the pattern of embryonic vasculogenic networks. Claudins are aberrantly expressed in aggressive breast cancer. However, the relationship between claudins and VM formation is not clear. We examined VM in two human breast cancer cell lines with different aggressive capabilities (MDA-MB-231 and MCF-7 cells) and one human umbilical vein endothelial cell line (HUVEC). Both HUVEC and MDA-MB-231 cells formed vascular channels in Matrigel cultures, while MCF-7 cells did not. Western blot analysis revealed a possible correlation between claudin-4 and -6 expression in breast cancer cell lines and tumor aggressiveness, with protein levels correlating with the ability to form vascular channels. Treatment of MDA-MB-231 and HUVEC cells with claudin-4 monoclonal antibodies completely inhibited the ability of cells to form vascular channels. Moreover, knockdown of claudin-4 by short hairpin RNA completely inhibited tubule formation in MDA-MB-231 cells. Overexpression of claudin-4 in MCF-7 cells induced formation of vascular channels. Immunocytochemistry revealed that membranous claudin-4 protein was significantly associated with vascular channel formation. Collectively, these results indicate that claudin-4 may play a critical role in VM in human breast cancer cells, opening new opportunities to improve aggressive breast cancer therapy.     

Integrin β1 is an essential factor in vasculogenic mimicry of human cancer cells

Vasculogenic mimicry (VM) formation by cancer cells is known to play a crucial role in tumor progression, but its detailed mechanism is unclear. In the present study, we focused on integrin β1 (ITGB1) and assessed the role of ITGB1 in VM formation. We used in vitro methods to seed cancer cells on Matrigel to evaluate the capability of VM formation. We carried out ITGB1 gene deletion using the CRISPR/Cas9 system, and these ITGB1‐knockout cells did not show a VM‐like network formation. Further, reintroduction of ITGB1 rescued VM‐like network formation in ITGB1‐knockout cells. In conclusion, ITGB1 is a critical factor in VM of human cancer cells, and inhibition of ITGB1 may be a novel therapeutic approach for malignant cancer.     

Microbial metabolite deoxycholic acid promotes vasculogenic mimicry formation in intestinal carcinogenesis

A high‐fat diet (HFD) leads to long‐term exposure to gut microbial metabolite secondary bile acids, such as deoxycholic acid (DCA), in the intestine, which is closely linked to colorectal cancer (CRC). Evidence reveals that vasculogenic mimicry (VM) is a critical event for the malignant transformation of cancer. Therefore, this study investigated the crucial roles of DCA in the regulation of VM and the progression of intestinal carcinogenesis. The effects of an HFD on VM formation and epithelial‐mesenchymal transition (EMT) in human CRC tissues were investigated. The fecal DCA level was detected in HFD‐treated Apc ^min/+ mice. Then the effects of DCA on VM formation, EMT, and vascular endothelial growth factor receptor 2 (VEGFR2) signaling were evaluated in vitro and in vivo. Here we demonstrated that compared with a normal diet, an HFD exacerbated VM formation and EMT in CRC patients. An HFD could alter the composition of the gut microbiota and significantly increase the fecal DCA level in Apc ^min/+ mice. More importantly, DCA promoted tumor cell proliferation, induced EMT, increased VM formation, and activated VEGFR2, which led to intestinal carcinogenesis. In addition, DCA enhanced the proliferation and migration of HCT‐116 cells, and induced EMT process and vitro tube formation. Furthermore, the silence of VEGFR2 reduced DCA‐induced EMT, VM formation, and migration. Collectively, our results indicated that microbial metabolite DCA promoted VM formation and EMT through VEGFR2 activation, which further exacerbated intestinal carcinogenesis.     

桃叶珊瑚苷调控RhoA/ROCK 信号通路对胃癌MGC803 细胞上皮间质转化和血管生成拟态的影响

目的：探究桃叶珊瑚苷（AU）调控RhoA/ROCK信号通路对胃癌MGC803细胞上皮间质转化（EMT）进程和血管生成拟态（VM）形成的影响。方法：常规培养人胃癌MGC803细胞,将其随机分为对照组、AU-L组（20μmol/L AU）、AU-M组（40μmol/L AU）、AU-H组（80μmol/L AU）、AU-H+RhoA激活剂水仙环素（Nar）组（AU-H+Nar组,80μmol/L AU+30μmol/L Nar）。采用CCK-8法、Transwell实验、细胞划痕实验分别检测不同浓度AU对细胞增殖、迁移和侵袭的影响,三维细胞培养法观察不同浓度AU对细胞体外VM管腔结构形成的影响,WB法检测AU对各组细胞RhoA、ROCK、VM与EMT相关蛋白表达的影响。结果：与对照组相比,AU-M组、AU-H组MGC803细胞增殖率（48、72 h时）、细胞迁移率、细胞侵袭数目、VM管腔结构数,以及RhoA、ROCK1、N-cadherin、vimentin、VE-cadherin的蛋白表达均显著降低（均P<0.05）,E-cadherin表达显著升高（P<0.05）;同时,使...     

扁蒴藤素调节Shh/Gli1信号通路对宫颈癌HeLa细胞增殖、凋亡和血管 生成拟态的影响

目的：探讨扁蒴藤素（Pris）调节Shh/Gli1信号通路对宫颈癌HeLa细胞增殖、凋亡和血管生成拟态（VM）的影响及其 机制。方法：采用MTT法检测不同浓度Pris对宫颈癌HeLa细胞增殖的抑制作用,以选取合适的干预浓度。将HeLa细胞分为对 照组、环巴胺组、Pris组、Pris+pc-NC组和Pris+pc-Shh组。采用MTT法、EdU法检测各组细胞的增殖能力,Transwell小室法、流式 细胞术检测各组细胞的迁移及侵袭能力和细胞凋亡率,体外血管生成实验观察VM形成情况,qPCR法检测各组细胞中Shh和 Gli1 mRNA表达水平,WB法检测细胞中血管内皮生长因子A（VEGF-A）、血管内皮钙黏素（VE-cadherin）、Ki-67、caspase-3及与 Shh/Gli1信号通路相关蛋白表达水平。结果：0.25~2.5 mmol/L的Pris 对HeLa细胞增殖均有显著抑制作用,选择1.5 mmol/L的 Pris进行后续实验。对照组细胞形成良好的管腔结构,与对照组相比,环巴胺组、Pris组和Pris+pc-NC组HeLa细胞管腔结构被明 显破坏,细胞增殖活力和增殖率、迁移及侵袭细胞数目、Shh和Gli1 mRNA、VEGF-A、VE-cadherin、Ki-67、Shh、Gli1蛋白表达均显 著降低（均P<0.05）,细胞凋亡率和caspase-3表达均显著升高（均P<0.05）;环巴胺组与Pris组HeLa细胞各项检测指标比较差异均 无统计学意义（均P>0.05）;与Pris+pc-NC组相比,Pris+pc-Shh组细胞管腔结构形成明显改善,细胞增殖活力和增殖率、迁移及侵 袭细胞数、Shh和Gli1 mRNA、VEGF-A、VE-cadherin、Ki-67、Shh、Gli1蛋白表达均显著升高（均P<0.05）,细胞凋亡率和caspase-3 表达均显著降低（均P<0.05）。结论：Pris抑制宫颈癌HeLa细胞的增殖、迁移与侵袭和VM的形成并促进细胞凋亡,可能与阻断 Shh/Gli1信号通路有关。     

DKK1 促进非小细胞肺癌线性程序性坏死和血管生成拟态形成*

目的:探讨非小细胞肺癌（non-small cell lung cancer ,NSCLC ）中DKK 1 影响线性程序性坏死（linearly patterned programmed cellnecrosis ,LPPCN ）和血管生成拟态（vasculogenic mimicry ,VM）的机制。方法:收集人NSCLC 标本173 例,H&E染色检测LP?PCN ,CD31/PAS 双染检测VM,免疫组织化学检测DKK 1 及相关蛋白表达,分析其临床病理意义及相互关系,进而裸鼠H 460-DKK 1 移植瘤体内验证研究假设。结果:NSCLC 中14.45%（25/ 173）存在VM,49.71%（86/ 173）具有LPPCN ,LPPCN （+）组25.6%（22/86）形成VM,二者均与分化差、TNM 分期晚、易复发转移和预后差相关。VM（+）组和LPPCN （+）组DKK 1 均高表达,均与VE-cad?herin、MMP-2、β -catenin 核表达及Twist1 正相关。H 460-DKK 1 移植瘤模型证实DKK 1 促进VM和LPPCN 及相关蛋白表达上调。结论:DKK 1 引起的β -catenin、Twist1 表达上调可促进NSCLC 中LPPCN 和VM形成。      

The role of sema4D in vasculogenic mimicry formation in non-small cell lung cancer and the underlying mechanisms

Vasculogenic mimicry (VM) is a special vascular pattern in malignant tumors, which is composed of highly aggressive tumor cells. This tumor cell-mediated blood supply pattern is closely associated with a poor prognosis in cancer patients. The interaction of axon guidance factor Sema4D and its high affinity receptor plexinB1 could activate small GTPase RhoA and its downstream ROCKs; this process has an active role in the migration of endothelial cells and tumor angiogenesis. Here, we have begun to uncover the role of this pathway in VM formation in non-small cell lung cancer (NSCLC). First, we confirmed this special form of vasculature in NSCLC tissues and found the existence of VM channels in tumor tissues was correlated with Sema4D expression. Further, we found that inhibition of Sema4D in the human NSCLC cells H1299 and HCC827 reduces VM formation both in vitro and in vivo. Moreover, we demonstrated that downregulating the expression of plexinB1 by siRNA expressing vectors and inhibiting the RhoA/ROCK signaling pathway using fasudil can reduce VM formation of H1299 and HCC827 cells. Finally, we found that suppression of Sema4D leads to less stress fibers and depleted the motility of H1299 and HCC827 cells. Collectively, our study implicates Sema4D plays an important role in the process of VM formation in NSCLC through activating the RhoA/ROCK pathway and regulating tumor cell plasticity and migration. Modulation of the Sema4D/plexinB1 and downstream RhoA/ROCK pathway may prevent the tumor blood supply through the VM pattern, which may eventually halt growth and metastasis of NSCLC.     

Application of functional vincristine plus dasatinib liposomes to deletion of vasculogenic mimicry channels in triple-negative breast cancer

Standard chemotherapy cannot eradicate triple-negative breast cancer (TNBC) while the residual cancer cells readily form the vasculogenic mimicry (VM) channels, which lead to the relapse of cancer after treatment. In this study, the functional vincristine plus dasatinib liposomes, modified by a targeting molecule DSPE-PEG₂₀₀₀-c(RGDyK), were fabricated to address this issue. The investigations were performed on TNBC MDA-MB-231 cells and MDA-MB-231 xenografts in nude mice. The liposomes exhibited the superior performances in the following aspects: the enhancement of cellular uptake via targeted action; the induction of apoptosis via activation of caspase 8, 9, and 3, increased expression of Bax, decreased expression of Mcl-1, and generation of reactive oxygen species (ROS); and the deletion of VM channels via inhibitions on the VM channel indicators, which consisted of vascular endothelial-cadherin (VE-Cad), focal adhesion kinase (FAK), phosphatidylinositide 3-kinase (PI3K), and matrix metallopeptidases (MMP-2, and MMP-9). Furthermore, the liposomes displayed the prolonged circulation time in the blood, the increased accumulation in tumor tissue, and the improved therapeutic efficacy along with deletion of VM channels in the TNBC-bearing mice. In conclusion, the nanostructured functional drug-loaded liposomes may provide a promising strategy for the treatment of invasive TNBC along with deletion of VM channels.     

Effect of grape seed proanthocyanidins on tumor vasculogenic mimicry in liver cancer xenograft mode

Objective： As a novel blood supply pattern, vasculogenic mimicry （VM） has attracted increasingly attention in recent years, which may partly compensate for the absence of feeding and facilitate tumor perfusion. However, anti-angiogenic drugs have little effect on VM. The grape seed proanthocyanidins （GSPs）, a kind of promising bioactive phytochemical, has shown anti-carcinogenesis and anti-angiogenic in several tumor models. However, GSPs regulation of VM and its possible mechanisms in a H22 hepatoma carcinoma model remain not clear. The aim of this study was to examine the effects of GSPs on proliferation and VM in a H22 hepatoma carcinoma model and to investigate the underlying mechanism. Methods： Seventy-five mice were divided into the control group and experimental groups treated with different concentration of GSPs. CD34-PAS dual staining was employed to identify the VM structure. The immunohistochemical staining for investigating the expression of VEGF, EphA2 and MMP-2 protein was performed. Results： Treatment of the H22 model with Endostar （4 mg/kg）, 50, 100, 200 mg/kg of the GSPs resulted in 6.87%, 17.81%, 27.43%, 53.52% inhibition in tumor growth, respectively. The mean weight of tumors were significantly lower in GSPs （100 mg/kg） and GSPs （200 mg/kg） groups than in the control group （all P 〈 0.01）. Similarly, compared with the control group, the number of VM channels were significantly reduced in GSPs （100 mg/kg） and GSPs （200 mg/kg） groups （all P 〈 0.01）. Immunohistochemistry showed significant decreases in the expression levels of VEGF, EphA2 and MMP-2 protein in GSPs （100 mg/kg） and GSPs （200 mg/kg） groups when compared with control group （all P 〈 0.001）. Conclusion： This is the first report providing evidence that GSPs inhibit the VM structure by regulation of the VEGF/EphA2/MMPs signaling pathway. Therefore, we concluded that GSPs has the potential of being a clinical anti-VM inhibitor.