Plastin 1 drives metastasis of colorectal cancer through the IQGAP1/Rac1/ERK pathway

Tumor metastasis is the dominant cause of death in colorectal cancer (CRC) patients, and it often involves dysregulation of various cytoskeletal proteins. Plastin 1 (PLS1) is an actin‐bundling protein that has been implicated in the structure of intestinal epithelial microvilli; however, its role in CRC metastasis has not yet been determined. In this study, we demonstrated that PLS1 is highly expressed in 33.3% (45/135) of CRC patients and is correlated with lymph node metastasis and poor survival. In in vitro and in vivo experiments, PLS1 induced the migration and invasion of CRC cells and the metastases to the liver and lung in mice. Moreover, the expressions of key factors for CRC metastases, matrix metalloproteinase (MMP) 9 and 2, were enhanced by PLS1, which was dependent on phosphorylating ERK1/2 activated by IQGAP1/Rac1 signaling. The connection between these signals and PLS1 was further confirmed in CRC tissues of patients and the metastatic nodules from a mouse model. These findings suggest that PLS1 promotes CRC metastasis through the IQGAP1/Rac1/ERK pathway. Targeting PLS1 may provide a potential approach to inhibit the metastasis of CRC cells.     

Angiogenin promotes colorectal cancer metastasis via tiRNA production

Metastasis of colorectal cancer (CRC) is the leading cause of CRC-associated mortality. Angiogenin (ANG), a member of the ribonuclease A superfamily, not only activates endothelial cells to induce tumor angiogenesis, but also targets tumor cells to promote cell survival, proliferation and/or migration. However, its clinical significance and underlying mechanism in CRC metastasis are still largely unknown. Here, we reported that ANG was upregulated in CRC tissues and associated with metastasis in CRC patients. We then revealed that ANG enhanced CRC growth and metastasis in both in vitro and in vivo systems. Intriguingly, we characterized a bunch of tRNA-derived stress-induced small RNAs (tiRNAs), produced through ANG cleavage, that was enriched in both CRC tumor tissues and highly metastatic cells, and functioned in ANG-promoted CRC metastasis. Moreover, higher level of a 5′-tiRNA from mature tRNA-Val (5′-tiRNA-Val) was observed in CRC patients and was correlated with tumor metastasis. Taken together, we propose that a novel ANG-tiRNAs-cell migration and invasion regulatory axis promotes CRC metastasis, which might be of potential target for CRC diagnosis and treatment.     

Moscatilin,a bibenzyl derivative from the India orchid Dendrobrium loddigesii, suppresses tumor angiogenesis and growth in vitro and in vivo

Attacking angiogenesis is considered an effective strategy for controls the expansion and metastasis of tumors and other related-diseases. The aim of this study was to assess the effects of moscatilin, a bibenzyl derivative, on VEGF and bFGF-induced angiogenesis in cultured human umbilical vein endothelial cells (HUVECs) in vitro and in vivo. Moscatilin significantly inhibited growth of lung cancer cell line A549 (NSCLC) and suppressed growth factor-induced neovascularization. In addition, VEGF- and bFGF-induced cell proliferation, migration, and tube formation of HUVECs was markedly inhibited by moscatilin. Western blotting analysis of cell signaling molecules indicated that moscatilin inhibited ERK1/2, Akt, and eNOS signaling pathways in HUVECs. These results suggest that inhibition of angiogenesis by moscatilin may be a major mechanism in cancer therapy.     

Progranulin promotes colorectal cancer proliferation and angiogenesis through TNFR2/Akt and ERK signaling pathways

Progranulin (PGRN) has been shown to be involved in the process of inflammation, wound healing, and cartilage development; and its role in the progression of breast and ovarian cancer is also well established. However, the expression status of PGRN in colorectal cancers (CRCs) and its molecular mechanisms responsible for tumorigenesis have not been addressed so far. Herein, we demonstrated that PGRN was highly expressed and had clinical relevance with CRCs since its overexpression was associated with advanced stages of CRCs, poorer patients’ prognosis, and increased expression of proliferation and angiogenesis markers. PGRN up-regulation significantly promoted the expression of Ki67 and vascular endothelial growth factor A (VEGF-A) as well as the growth rate in CRC cell lines, while PGRN down-regulation had the opposite effects. Strikingly, PGRN derived from CRCs could directly induce proliferation, migration, tubule formation, as well as VEGF-A expression in human umbilical vein endothelial cells (HUVECs). Furthermore, we provided mechanistic evidences that the regulation of Ki67 and VEGF-A expression by PGRN was mediated by tumor necrosis factor receptor 2 (TNFR2)/Akt and the ERK signaling pathways in both CRC cells and HUVECs. Taken together, these findings suggested that PGRN could promote proliferation and angiogenesis through TNFR2/Akt and ERK signaling pathways in CRCs, providing the new insight into the mechanism of PGRN in tumor proliferation and angiogenesis.     

Upregulation of adipocyte enhancer‐binding protein 1 in endothelial cells promotes tumor angiogenesis in colorectal cancer

Tumor angiogenesis is an important therapeutic target in colorectal cancer (CRC). We aimed to identify novel genes associated with angiogenesis in CRC. Using RNA sequencing analysis in normal and tumor endothelial cells (TECs) isolated from primary CRC tissues, we detected frequent upregulation of adipocyte enhancer‐binding protein 1 (AEBP1) in TECs. Immunohistochemical analysis revealed that AEBP1 is upregulated in TECs and stromal cells in CRC tissues. Quantitative RT‐PCR analysis showed that there is little or no AEBP1 expression in CRC cell lines, but that AEBP1 is well expressed in vascular endothelial cells. Levels of AEBP1 expression in Human umbilical vein endothelial cells (HUVECs) were upregulated by tumor conditioned medium derived from CRC cells or by direct coculture with CRC cells. Knockdown of AEBP1 suppressed proliferation, migration, and in vitro tube formation by HUVECs. In xenograft experiments, AEBP1 knockdown suppressed tumorigenesis and microvessel formation. Depletion of AEBP1 in HUVECs downregulated a series of genes associated with angiogenesis or endothelial function, including aquaporin 1 (AQP1) and periostin (POSTN), suggesting that AEBP1 might promote angiogenesis through regulation of those genes. These results suggest that upregulation of AEBP1 contributes to tumor angiogenesis in CRC, which makes AEBP1 a potentially useful therapeutic target.     

Targeting the ERK pathway reduces liver metastasis of Smad4-inactivated colorectal cancer

Transforming growth factor β (TGF-β)/Smad signaling is involved in colorectal carcinoma (CRC) development and progression. The frequent loss of SMAD4 is associated with liver metastasis and poor prognosis of CRC, but the underlying mechanism remains elusive. This study aimed to elucidate the role of Smad-independent TGF-β signaling in CRC metastasis. Immunohistochemistry showed that Smad4 level was negatively correlated with TNM stage and phospho-ERK level in human CRCs and liver metastasis samples. Knockdown of Smad4 in CT26 and HCT116 cells activated ERK pathway, altered the expression of MMP2 and COX-2, promoted cell motility, migration, and invasion in vitro, enhanced metastasis, and shortened the survival of metastatic tumor-bearing mice. MEK inhibitor U0126 and GSK1120212 inhibited the motility, migration, and invasion of Smad4 knockdown cells, inhibited metastasis, and prolonged the survival of metastatic tumor-bearing mice. Furthermore, MEK inhibitor could reverse the changes of phospho-ERK, MMP2, and COX-2 levels. In conclusion, our results indicate that ERK pathway plays a key oncogenic role in CRC with SMAD4 inactivation mutations, and implicate ERK as a potential therapeutic target for CRC liver metastasis.     

Targeting EGFL7 Expression through RNA Interference Suppresses Renal Cell Carcinoma Growth by Inhibiting Angiogenesis

Renal cell carcinoma (RCC) is the most lethal of all urological cancers and tumor angiogenesis is closely relatedwith its growth, invasion, and metastasis. Recent studies have suggested that epidermal growth factor-like domainmultiple 7 (EGFL7) is overexpressed by many tumors, such as colorectal cancer and hepatocellular carcinoma;it is also correlated with progression, metastasis, and a poor prognosis. However, the role of EGFL7 in RCC isnot clear. In this study, we examined how EGFL7 contributes to the growth of RCC using a co-culture systemin vitro and a xenograft model in vivo. Downregulated EGFL7 expression in RCC cells affected the migrationand tubule formation of HMEC-1 cells, but not their growth and apoptosis in vitro. The level of focal adhesionkinase (FAK) phosphorylation in HMEC-1 cells decreased significantly when co-cultured with 786-0/iEGFL7cells compared with 786-0 cells. After adding rhEGFL7, the level of FAK phosphorylation in HMEC-1 cells wassignificantly elevated compared with phosphate-buffered saline (PBS) control. However, FAK phosphorylationwas abrogated by EGFR inhibition. The average size of RCC local tumors in the 786-0/iEGFL7 group wasnoticeably smaller than those in the 786-0 cell group and their vascular density was also significantly decreased.These data suggest that EGFL7 has an important function in the growth of RCC by facilitating angiogenesis.     

FGFR3 promotes angiogenesis-dependent metastasis of hepatocellular carcinoma via facilitating MCP-1-mediated vascular formation

The biological role of fibroblast growth factor receptor 3 (FGFR3) in tumor angiogenesis of hepatocellular carcinoma (HCC) has not been discussed before. Our previous work had indicated FGFR3 was overexpressed in HCC, and silencing FGFR3 in Hu7 cells could regulate tumorigenesis via down-regulating the phosphorylation level of key members of classic signaling pathways including ERK and AKT. In the present work, we explored the role of FGFR3 in angiogenesis-dependent metastasis by using SMMC-7721 and QGY-7703 stable cell lines. Our results indicated FGFR3 could regulate in vitro cell migration ability and in vivo lung metastasis ability of HCC, which was in accordance with increased angiogenesis ability in vitro and in vivo. Using the supernatant from SMMC-7721/FGFR3 cells, we conducted a human angiogenesis protein microarray including 43 angiogenesis factors and found that FGFR3 modulated angiogenesis and metastasis of HCC mainly by promoting the protein level of monocyte chemotactic protein 1 (MCP-1). Silencing FGFR3 by short hairpin RNA (shRNA) could reduce MCP-1 level in lysates and supernatant of QGY-7703 cells and SMMC-7721 cells. Silencing MCP-1 in QGY-7703 or SMMC-7721 cells could induce similar phenotypes compared with silencing FGFR3. Our results suggested FGFR3 promoted metastasis potential of HCC, at least partially if not all, via facilitating MCP-1-mediated angiogenesis, in addition to previously found cell growth and metastasis. MCP-1, a key medium between HCC cells and HUVECs, might be a novel anti-vascular target in HCC.     

Expression of Epidermal Growth Factor-like Domain 7 is Increased by Transcatheter Arterial Embolization of Liver Tumors

Background: Epidermal growth factor-like domain multiple 7 (EGFL7), recently identified as a secretedprotein regulated by oxygen exposure, plays a critical role in promoting metastasis of hepatocellular carcinoma(HCC). Transcatheter arterial embolization (TAE) is widely used for treatment of HCC, resulting in hypoxia intumors and surrounding liver tissues. Accordingly, we proposed the hypothesis that there could be a relationshipbetween expression of EGFL7 and response to TAE. Materials and Methods: We established a rabbit VX2liver tumor model using percutaneous puncture technique guided by computed tomography. TAE and shamembolization were performed and the results were confirmed by MRI 3 weeks after inoculation. We investigatedthe EGFL7 expression of the two groups at 6h and 3 days after intervention by means of immunohistochemistryand Western blotting. Results: Immunohistochemical staining demonstrated that the levels of EGFL7 proteinsignificantly increased in the TAE-treated tumors compared with the control group at 6 hours (P=0.031) and 3days (P=0.020) after intervention. Meanwhile, the relative EGFL7 protein detected in TAE group also up-regulatedcompared with the control group at 6 hours (P=0.020) and 3 days (P=0.024) after intervention. Conclusions:This study reveals an increase of EGFL7 expression in rabbit VX2 liver tumors after TAE. The role of EGFL7in HCC, especially its biological behavior after TAE, needs further investigation.     

细胞骨架相关蛋白4在肾透明细胞癌中的表达及其临床意义

目的 探讨细胞骨架相关蛋白4(cytoskeleton-associated protein 4,CKAP4)在肾透明细胞癌(clear cell renal cell carcinoma,ccRCC)中的表达及其临床意义。方法 收集237例ccRCC患者的癌组织和癌旁组织,采用免疫组化法检测CKAP4蛋白的表达水平,采用χ²检验比较不同CKAP4蛋白表达水平患者的临床病理特征,Kaplan-Meier法和Log-rank检验分析不同CKAP4表达水平患者总生存期(overall survival,OS)和无进展生存期(progression-free survival,PFS)的差异,Cox比例风险回归分析影响ccRCC患者术后OS和PFS的因素。结果 ccRCC组织中CKAP4蛋白的高表达率显著高于癌旁组织(54.0%vs 23.6%,χ²=46.050,P<0.001)。Fuhrman分级高、肿瘤浸润深、有淋巴结转移、远处转移以及TNM分期高的ccRCC患者癌组织中CKAP4蛋白高表达(均P<0.05)。CKAP4蛋白高表达...     

ING4对缺氧状态下血管内皮细胞增殖、迁移及HIF-1α表达的抑制作用

目的:在缺氧状态下,观察ING4对血管内皮细胞增殖、迁移及血管形成相关因子VEGF、MMP-2、MMP-9活性的影响,并探讨ING4对HIF-1α的调控作用。方法:人脐静脉内皮细胞HUVECs体外培养,构建ING4质粒及干扰RNA转染至HUVECs,应用CoCl2处理细胞模拟缺氧环境,MTT法检测细胞增殖,Transwell实验检测细胞迁移,Real time PCR及Western blot实验检测血管形成相关因子VEGF、MMP-2、MMP-9、HIF-1α及其靶基因AK3的mRNA及蛋白表达水平。结果:MTT及Transwell实验结果显示在缺氧状态下,转染ING4质粒能够抑制人脐静脉内皮细胞株HUVECs的增殖及迁移,而转染ING4 siRNA则能够促进HUVECs增殖及迁移,Real time PCR及Western blot实验结果显示转染ING4质粒能够抑制血管形成相关因子VEGF、MMP-2、MMP-9 mRNA及蛋白的表达水平,并且能够抑制HIF-1α及其靶基因AK3的表达水平。结论:在缺氧状态下,ING4能够通过抑制血管内皮细胞的增殖及迁移能力,下调血管形成相关因子VEGF、MMP-2、MMP-9的表达水平,进而抑制新血管形成,其机制可能与ING4能够下调缺氧状态下HIF-1α及其靶基因AK3的表达水平有关。     

CXCL16/CXCR6 chemokine signaling mediates breast cancer progression by pERK1/2-dependent mechanisms

Our previous studies demonstrate that CXCL6/CXCR6 chemokine axis induces prostate cancer progression by the AKT/mTOR signaling pathway; however, its role and mechanisms underlying invasiveness and metastasis of breast cancer are yet to be elucidated. In this investigation, CXCR6 protein expression was examined using high-density tissue microarrays and immunohistochemistry. Expression of CXCR6 shows a higher epithelial staining in breast cancer nest site and metastatic lymph node than the normal breast tissue, suggesting that CXCR6 may be involved in breast cancer (BC) development. In vitro and in vivo experiments indicate that overexpression of CXCR6 in BC cells has a marked effect on increasing cell migration, invasion and metastasis. In contrast, reduction of CXCR6 expression by shRNAs in these cells greatly reduce its invasion and metastasis ability. Mechanistic analyses show that CXCL16/CXCR6 chemokine axis is capable of modulating activation of RhoA through activating ERK1/2 signaling pathway, which then inhibits the activity of cofilin, thereby enhancing the stability of F-actin, responsible for invasiveness and metastasis of BC.Taken together, our data shows for the first time that the CXCR6 / ERK1/2/ RhoA / cofilin /F-actin pathway plays a central role in the development of BC. Targeting the signaling pathway may prove beneficial to prevent metastasis and provide a more effective therapeutic strategy for BC.     

Anti-tumor effect of lentivirus-mediated gene transfer of alphastatin on human glioma

Alphastatin, an endogenous angiogenesis inhibitor, has recently been used as an anticancer agent in several tumor models. This study was to investigate whether local sustained long-term expression of alphastatin could serve to diminish tumor growth of a human xenograft glioma model. We found that the recombinant alphastatin lentiviruses were able to stably infect HUVECs, and infected HUVECs could sustainably secrete alphastatin, which exhibited potent inhibitory effects on HUVECs migration, differentiation but not proliferation induced by vascular endothelial growth factor (VEGF) or basic fibroblast growth factor(bFGF). And the expression of secreted protein alphastatin markedly decreased tumor vascularization and inhibited tumor growth. Additionally, alphastatin inhibited VEGF- or bFGF-induced initial stage of angiogenesis by reducing JNk and ERK phosphorylation in vitro. Taken together, these data demonstrate that secreted protein alphastatin inhibits VEGF- or bFGF-induced angiogenesis by suppressing JNK and ERK kinases activation pathways in HUVECs, and markedly inhibits tumor angiogenesis in vivo. Consequently lentivirus-mediated gene transfer might represent an effective strategy for expression of alphastatin to achieve inhibition of human malignant glioma proliferation and tumor progression.     

RFC5, regulated by circ_0038985/miR-3614-5p,functions as an oncogene in the progression of colorectal cancer

Replication factor C 5 (RFC5) is involved in a variety of biological functions of cancer. However, the expression pattern of RFC5 and the underlying mechanisms in colorectal cancer (CRC) remain elusive. Here, we show that RFC5 is significantly upregulated in CRC tissues and cells. Patients with CRC and increased RFC5 levels have an unfavorable prognosis. RFC5 can promote the proliferation, migration, and invasion of CRC cells and inhibit the apoptosis of CRC cells. Additionally, upstream of RFC5, we constructed the competing endogenous RNA network and confirmed that RFC5 in this network was inhibited by miR-3614-5p by directly targeting its 3′-untranslated regions. We verified that circ_0038985, which is positively correlated with RFC5, directly targeted miR-3614-5p. Overexpression of circ_0038985 promoted CRC cell migration and invasion, and these effects were partially reversed by the reintroduction of miR-3614-5p. Moreover, we found that RFC5 may promote the vascular endothelial growth factor A (VEGFa)/vascular endothelial growth factor receptor 2 (VEGFR2)/extracellular signal-regulated protein kinase (ERK) pathway. The knockdown of RFC5 reduced CRC tumorigenesis in vivo. Collectively, these data demonstrate that the circ_0038985/miR-3614-5p/RFC5 axis plays a critical role in the progression of CRC, and RFC5 may promote CRC progression by affecting the VEGFa/VEGFR2/ERK pathway.     

外泌体中circ_0044366调控肿瘤相关成纤维细胞MMP2的表达促进结直肠癌转移的机制研究

背景与目的:结直肠癌(colorectal cancer,CRC)是全球癌症死亡的主要原因之一,大量研究发现,环状RNA(circular RNA,circRNA)作为miRNA分子海绵参与CRC细胞的增殖、迁移、侵袭和凋亡等病理生理学过程,circRNA/miRNA/靶基因/靶蛋白轴可能是CRC发生的重要原因之一.探...     

Interleukin-8 promotes cell migration through integrin αvβ6 upregulation in colorectal cancer

Colorectal cancer (CRC), which is notorious for high morbidity and mortality around the world, shows a predilection for metastasis to liver. Interleukin-8 (IL-8), a chemokine with a defining CXC amino acid motif, has been reported to promote CRC cell migration and is associated with poor prognosis of CRC. However, the underlying molecular mechanism of IL-8-mediated migration remains obscure. In this study, we first demonstrated the cross talk between IL-8 and integrin αvβ6. We analyzed 139 human CRC samples, and found that the immunohistochemical expression of αvβ6 was significantly correlated with expression of IL-8. Furthermore, IL-8 increased the migration through integrin αvβ6 in human CRC cells, and both CXCR1 and CXCR2 were primarily involved during the process. IL-8 upregulated αvβ6 expression in a dose-dependent manner through activation of ERK and Ets-1 signaling pathway. Taken together, our results indicated that IL-8 enhances the migration of CRC cells by increasing αvβ6 integrin expression through the ERK/Ets-1 pathway. Targeting integrin αvβ6 in IL-8 expressing tumors might be a potential therapeutic strategy for CRC patients.     

二氢杨梅素通过ERK/VEGFA/VEGFR2通路对体内外胃癌血管生成的影响

背景与目的：二氢杨梅素（dihydromyricetin,DHM）通过抑制细胞周期、促进细胞凋亡和抑制新生血管生成等机制发挥抗肿瘤作用。探讨DHM对胃癌的抗癌作用,并研究其可能作用机制。方法：不同剂量DHM作用体外人脐静脉内皮细胞（human umbilical vein endothelial cells, HUVEC）和胃癌细胞系MKN28后,采用细胞计数试剂盒（cell counting kit-8,CCK-8）法筛选无毒剂量的DHM,Transwell小室实验测定细胞侵袭;小管形成实验测定HUVEC体外血管生成能力;采用蛋白质印迹法（Western blot）检测血管内皮生长因子A（vascular endothelial growth factor A, VEGFA）、phospho-血管内皮生长因子受体（vascular endothelial growth factor receptor, VEGFR）2、细胞外信号调节激酶（extracellular signal-regulated kinase,ERK）、phospho-c-Jun氨基端激酶（c-Jun NH2-terminal kinase,JNK）和phospho-p38表达水平;基质胶塞实验测定DHM对体内血管形成的影响;通过皮下肿瘤细胞注射法建立MKN28裸鼠异种移植瘤模型,观察DHM给药对体内胃癌生长的影响,Ki-67增殖指数及CD34、VEGFA在移植瘤组织中的表达通过免疫组织化学法测定。结果：（0.5~2.5 μmol/L）的DHM对HUVEC和MKN28细胞生长无明显抑制作用,该浓度范围的DHM呈浓度依赖性地抑制HUVEC侵袭和小管形成,VEGFA（20 μg/L）可明显逆转DHM对HUVEC侵袭和小管形成的抑制效果;DHM处理可导致MKN28细胞中VEGFA和phospho-ERK表达呈剂量依赖性下降,而对p-p38和p-JNK的表达无明显影响;DHM处理可导致HUVEC中phospho-VEGFR2表达呈剂量依赖性下降;低剂量（30 mg/kg）DHM对裸鼠无明显的不良反应,但可抑制体内血管形成,同时有效延缓体内胃癌生长,体内肿瘤组织中的CD34和VEGFA的表达受到DHM的显著下调作用,但低剂量DHM对Ki-67无明显作用。结论：低剂量的DHM可有效抑制体内外血管生成和体内胃癌生长,该作用至少部分是通过ERK/VEGFA/VEGFR2信号通路来实现的。