结直肠腺癌血管生成拟态的形态学观察及其临床病理意义

目的:研究结直肠腺癌中是否存在血管生成拟态(vasculogenic mimicry,VM)及其临床病理意义。方法:收集156例结直肠腺癌组织学标本及其临床资料,利用CD34和PAS双重染色,观察是否存在VM,分析VM的形成与结直肠腺癌临床病理指标的关系。结果:156例结直肠腺癌中有31例存在VM,阳性率为19.87%,VM的形成与肿瘤大小无关,在低分化组、伴有血管及神经侵犯组、伴有淋巴结转移组,VM的阳性率比在中分化和高分化组、无血管及神经侵犯组、无淋巴结转移组高,差异有统计学意义（P＜0.05）。结论:结直肠腺癌中存在VM,VM形成与结直肠腺癌的侵袭性或远处转移相关。     

仿血管通道结构在大鼠和裸鼠骨肉瘤组织微血管形成中的作用

目的:研究肿瘤仿血管通道在骨肉瘤裸鼠移植瘤组织中的结构特点及其在肿瘤微血管形成中的作用。方法:建立荷人骨肉瘤MG-63细胞裸鼠移植瘤和荷骨肉瘤UMR106细胞大鼠移植瘤模型,以CD34、CD147免疫荧光和GFP荧光法观察移植瘤组织中肿瘤仿血管通道的结构特点及其与肿瘤微血管的关系。结果:骨肉瘤MG-63细胞移植瘤和UMR106细胞移植瘤组织CD147染色均呈阳性,血管内皮细胞CD34染色均阳性。肿瘤仿血管通道主要位于移植瘤组织中心区域,主要由骨肉瘤细胞构成,是具有功能的类血管通道;肿瘤微血管主要位于移植瘤周边区域,主要由宿主组织的血管内皮细胞组成,是与肿瘤仿血管通道存在直接相通的管道样结构。结论:大鼠和裸鼠骨肉瘤细胞移植瘤组织中存在的肿瘤仿血管通道具有类血管功能,并与来源于宿主组织的肿瘤微血管相互联通,诱导肿瘤微血管侵入肿瘤内部生长。     

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.     

Frizzled 2‐induced epithelial‐mesenchymal transition correlates with vasculogenic mimicry,stemness, and Hippo signaling in hepatocellular carcinoma

Prior observation has indicated that Frizzled 2 (FZD2)‐induced epithelial‐mesenchymal transition (EMT) could be a key step in metastasis and early recurrence of hepatocellular carcinoma (HCC). However, the mechanism underlying tumor development and progression due to aberrant FZD2 expression is poorly defined. Here, we provide evidence that FZD2 is a driver for EMT, cancer stem cell properties, and vasculogenic mimicry (VM) in HCC. We found that FZD2 was highly expressed in two cohorts of Chinese hepatitis B virus‐related HCC patients, and that high FZD2 expression was associated with poor prognosis. Concerning the mechanism, gain‐ and loss‐of‐function experiments showed the oncogenic action of FZD2 in HCC cell proliferation, apoptosis, migration, and invasion. Further investigations in vitro and in vivo suggested that FZD2 promotes the EMT process, enhances stem‐like properties, and confers VM capacity to HCC cells. Notably, integrative RNA sequencing analysis of FZD2‐knockdown cells indicated the enrichment of Hippo signaling pathway. Taken together, our data suggest for the first time that FZD2 could promote clinically relevant EMT, CD44⁺ stem‐like properties, and the VM phenotype in HCC involving a potential Hippo signaling pathway‐dependent mechanism, and should be considered as a promising therapeutic target for the treatment of HCC.     

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.     

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。

     

马钱子碱抑制乳腺癌细胞体外血管生成拟态的形成及其可能机制研究

背景与目的：乳腺癌等多种高度侵袭性的肿瘤中存在血管生成拟态（vasculogenic mimicry, VM）现象,可能导致传统的抗血管治疗效果不理想。马钱子碱是马钱子的主要生物碱单体,具有潜在的抗血管生成作用。本研究旨在探讨马钱子碱对人乳腺癌细胞系MDA-MB-231体外形成血管生成拟态的影响及其可能的作用机制。方法：采用MTT法测定马钱子碱对人乳腺癌细胞增殖的抑制作用;应用Annexin Ⅴ-FITC/碘化丙啶（propidium iodide,PI）双染流式细胞术检测细胞凋亡情况,以Hoechst 33342/PI（HO/PI）染色进行细胞死亡检测。在Matrigel基质胶上三维培养人乳腺癌细胞,研究在体外是否可以形成血管生成拟态,并观察马钱子碱对这一过程的影响。采用蛋白质印迹法（Western blot）检测血管生成拟态相关标志分子表达的变化,如血管内皮生长因子A（vascular endothelial growth factor-A,VEGF-A）、血管内皮钙黏蛋白（vascular endothelial cadherin,VE-cadherin）、EphA2和基质金属蛋白酶（matrix metalloprotein,MMP）。结果：马钱子碱可有效地抑制乳腺癌细胞增殖,且随药物浓度增加,细胞凋亡与坏死比例增大,细胞死亡率升高;马钱子碱可抑制乳腺癌细胞血管生成拟态的形成,呈剂量依赖性;马钱子碱处理后血管生成拟态标志蛋白（VEGF/VE-cadherin/EphA2/MMP-9/MMP-2）的表达水平明显下调。结论：马钱子碱可以抑制乳腺癌细胞系MDA-MB-231增殖,诱导其凋亡,并抑制其体外血管生成拟态的形成,其机制可能与VEGF/VE-cadherin/EphA2/MMP-9/MMP-2的表达下调有关。     

Curcumin suppresses invasiveness and vasculogenic mimicry of squamous cell carcinoma of the larynx through the inhibition of JAK-2/STAT-3 signaling pathway

To determine the role of JAK-2/STAT-3 signaling pathway in invasion and vasculogenic mimicry of laryngeal squamous cell carcinoma. HEp-2 cells were treated with 1 or 10 μmol/L curcumin and AG490 (the inhibitor of JAK-2) for 48 h, the invasion and vasculogenic mimicry of tumor cells were tested with Transwell chamber test and tube formation experiment. RT-PCR was used to measure the expression of MMP-2 and VEGF. Western blot assay was employed to determine the expression of JAK-2, STAT3, p-STAT3, MMP-2 and VEGF. Compared to control group，there were less tumor cells permeating membrane and less formed tubes after curcumin or AG490 treatment, RT-PCR showed that the expression of MMP-2 and VEGF at mRNA level were decreased (P < 0.01). Western blotting indicated that the expression of JAK-2, p-STAT3, MMP-2 and VEGF at protein levels were decreased (P < 0.01), while that of STAT-3 protein had no difference among each group (P > 0.05). Immunofluorescence staining demonstrated that the expression of eNOS was down-regulated (P < 0.01). Curcumin and AG490 significantly inhibits invasion and vasculogenic mimicry of laryngeal squamous cell carcinoma in vitro, and JAK-2/STAT-3 signaling pathway promotes above processes.     

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.     

Metronomic gemcitabine suppresses tumour growth,improves perfusion,and reduces hypoxia in human pancreatic ductal adenocarcinoma

Background:

The current standard of care for pancreatic cancer is weekly gemcitabine administered for 3 of 4 weeks with a 1-week break between treatment cycles. Maximum tolerated dose (MTD)-driven regimens as such are often associated with toxicities. Recent studies demonstrated that frequent dosing of chemotherapeutic drugs at relatively lower doses in metronomic regimens also confers anti-tumour activity but with fewer side effects.

Methods:

Herein, we evaluated the anti-tumour efficacy of metronomic vs MTD gemcitabine, and investigated their effects on the tumour microenvironment in two human pancreatic cancer xenografts established from two different patients.

Results:

Metronomic and MTD gemcitabine significantly reduced tumour volume in both xenografts. However, K^trans values were higher in metronomic gemcitabine-treated tumours than in their MTD-treated counterparts, suggesting better tissue perfusion in the former. These data were further supported by tumour-mapping studies showing prominent decreases in hypoxia after metronomic gemcitabine treatment. Metronomic gemcitabine also significantly increased apoptosis in cancer-associated fibroblasts and induced greater reductions in the tumour levels of multiple pro-angiogenic factors, including EGF, IL-1α, IL-8, ICAM-1, and VCAM-1.

Conclusion:

Metronomic dosing of gemcitabine is active in pancreatic cancer and is accompanied by pronounced changes in the tumour microenvironment.     

Overexpression of receptor tyrosine kinase Axl promotes tumor cell invasion and survival in pancreatic ductal adenocarcinoma

BACKGROUND:

The receptor tyrosine kinase Axl has been reported to be overexpressed in a variety of human cancers. Although previous studies have identified the role of Axl in the transformation, proliferation, survival, and invasion in cancers, the expression and functions of Axl in pancreatic cancer have not been studied in detail.

METHODS:

The expression of Axl protein in 12 pancreatic cancer cell lines and 54 patient samples of stage II pancreatic ductal adenocarcinoma (PDA) and their paired non‐neoplastic pancreatic tissue samples were examined. Using univariate and multivariate analysis, Axl expression was correlated with survival and other clinicopathologic features. To examine Axl functions in PDA, the effects of Axl knockdown on the invasion ability and radiation‐induced apoptosis in PDA cell lines were measured.

RESULTS:

Axl was overexpressed in 38 of 54 (70%) stage II PDA samples and 9 of 12 (75%) PDA cell lines. Axl overexpression was associated with higher frequencies of distant metastasis and poor overall and recurrence‐free survivals (P = .03 and P = .04, respectively) independent of tumor size and stage or lymph node status in patients with stage II PDA. Knockdown of Axl expression in PDA cells abolished Gas6‐mediated Akt activation, decreased invasion, and increased radiation‐induced PARP cleavage and the percentage of apoptosis.

CONCLUSIONS:

This study showed that Gas6 and Axl are frequently overexpressed in PDA cells and are associated with a poor prognosis in patients with stage II PDA. Axl promotes the invasion and survival of PDA cells. Therefore, targeting the Axl signaling pathway may represent a new approach to the treatment of PDA. Cancer 2011. © 2010 American Cancer Society.     

Cilengitide downmodulates invasiveness and vasculogenic mimicry of neuropilin 1 expressing melanoma cells through the inhibition of αvβ5 integrin

During melanoma progression, tumour cells show increased adhesiveness to the vascular wall, invade the extracellular matrix (ECM) and frequently form functional channels similar to vascular vessels (vasculogenic mimicry). These properties are mainly mediated by the interaction of integrins with ECM components. Since we had previously identified neuropilin 1 (NRP‐1), a coreceptor of vascular endothelial growth factor A (VEGF‐A), as an important determinant of melanoma aggressiveness, aims of this study were to identify the specific integrins involved in the highly invasive phenotype of NRP‐1 expressing cells and to investigate their role as targets to counteract melanoma progression. Melanoma aggressiveness was evaluated in vitro as cell ability to migrate through an ECM layer and to form tubule‐like structures using transfected cells. Integrins relevant to these processes were identified using specific blocking antibodies. The αvβ5 integrin was found to be responsible for about 80% of the capability of NRP‐1 expressing cells to adhere on vitronectin. In these cells αvβ5 expression level was twice higher than in low‐invasive control cells and contributed to the ability of melanoma cells to form tubule‐like structures on matrigel. Cilengitide, a potent inhibitor of αν integrins activation, reduced ECM invasion, vasculogenic mimicry and secretion of VEGF‐A and metalloproteinase 9 by melanoma cells. In conclusion, we demonstrated that ανβ5 integrin is involved in the highly aggressive phenotype of melanoma cells expressing NRP‐1. Moreover, we identified a novel mechanism that contributes to the antimelanoma activity of the αv integrin inhibitor cilengitide based on the inhibition of vasculogenic mimicry.     

FXYD3 is overexpressed in pancreatic ductal adenocarcinoma and influences pancreatic cancer cell growth

The expression and localization of FXYD domain containing ion transport regulator 3 (FXYD3), a transmembrane protein that acts as a chloride channel or chloride channel regulator, was analyzed in pancreatic tissues derived from donors and patients suffering from chronic pancreatitis (CP) or pancreatic ductal adenocarcinoma (PDAC) as well as in pancreatic cancer cells using QRT-PCR, laser-capture microdissection and microarray analysis, in situ hybridization and immunohistochemistry. FXYD3 antisense expressing T3M4 pancreatic cancer cells were generated and compared to control cells using anchorage-dependent and independent growth assays, and xenotransplantation into nude mice. FXYD3 mRNA levels were 3.4-fold increased in PDAC tissues compared to donor specimens (p = 0.006), and 3.9-fold increased in microdissected cancer cells compared to normal pancreatic ductal cells (p = 0.02). FXYD3 was localized in the tubular complexes and PanIN lesions of both CP and PDAC, as well as in pancreatic cancer cells. Downregulation of FXYD3 by stable antisense transfection increased significantly the doubling time of T3M4 pancreatic cancer cells from 44 +/- 2 hr to 55 +/- 12 hr (p = 0.02). Nude mice transplanted with antisense transfected cells displayed a significant increase in tumor doubling time from 3.3 days +/- 1.0 to 4.3 days +/- 0.43 (p = 0.058). Anchorage-independent growth and sensitivity to 5-fluorouracil, gemcitabine and cisplatin as well as to MgCl(2) were not dependent on the level of FXYD3 expression. In conclusion, overexpression of FXYD3 in pancreatic cancer may contribute to the proliferative activity of this malignancy.     

Post-therapy pathologic stage and survival in patients with pancreatic ductal adenocarcinoma treated with neoadjuvant chemoradiation

BACKGROUND:

Neoadjuvant chemoradiation before surgery is an emerging treatment modality for pancreatic ductal adenocarcinoma (PDAC). However, analysis of prognostic factors is limited for patients with PDAC treated with neoadjuvant chemoradiation and pancreaticoduodenectomy (PD).

METHODS:

The study population was comprised of 240 consecutive patients with PDAC who received neoadjuvant chemoradiation and PD and was compared with 60 patients who had no neoadjuvant therapy between 1999 and 2007. Clinicopathologic features were correlated with disease‐free survival (DFS) and overall survival (OS).

RESULTS:

Among the 240 treated patients, the 1‐year and 3‐year DFS rates were 52% and 32%, with a median DFS of 15.1 months. The 1‐year and 3‐year OS rates were 95% and 47%, with a median OS of 33.5 months. By univariate analysis, DFS was associated with age, post‐therapy tumor stage (ypT), lymph node status (ypN), number of positive lymph nodes, and American Joint Committee on Cancer (AJCC) stage, whereas OS was associated with intraoperative blood loss, margin status, ypT, ypN, number of positive lymph nodes, and AJCC stage. By multivariate analysis, DFS was independently associated with age, number of positive lymph nodes, and AJCC stage, and OS was independently associated with differentiation, margin status, number of positive lymph nodes, and AJCC stage. In addition, the treated patients had better OS and lower frequency of lymph node metastasis than those who had no neoadjuvant therapy.

CONCLUSIONS:

In patients with PDAC who received neoadjuvant chemoradiation and subsequent PD, post‐therapy pathologic AJCC stage and number of positive lymph nodes are independent prognostic factors. Cancer 2012. © 2011 American Cancer Society.     

Oxidative inhibition of Hsp90 disrupts the super‐chaperone complex and attenuates pancreatic adenocarcinoma in vitro and in vivo

Pancreatic cancer is almost always fatal, in part because of its delayed diagnosis, poor prognosis, rapid progression and chemoresistance. Oncogenic proteins are stabilized by the Hsp90, making it a potential therapeutic target. We investigated the oxidative stress‐mediated dysfunction of Hsp90 and the hindrance of its chaperonic activity by a carbazole alkaloid, mahanine, as a strategic therapeutic in pancreatic cancer. Mahanine exhibited antiproliferative activity against several pancreatic cancer cell lines through apoptosis. It induced early accumulation of reactive oxygen species (ROS) leading to thiol oxidation, aggregation and dysfunction of Hsp90 in MIAPaCa‐2. N‐acetyl‐L ‐cysteine prevented mahanine‐induced ROS accumulation, aggregation of Hsp90, degradation of client proteins and cell death. Mahanine disrupted Hsp90‐Cdc37 complex in MIAPaCa‐2 as a consequence of ROS generation. Client proteins were restored by MG132, suggesting a possible role of ubiquitinylated protein degradation pathway. Surface plasmon resonance study demonstrated that the rate of interaction of mahanine with recombinant Hsp90 is in the range of seconds. Molecular dynamics simulation showed its weak interactions with Hsp90. However, no disruption of the Hsp90‐Cdc37 complex was observed at an early time point, thus ruling out that mahanine directly disrupts the complex. It did not impede the ATP binding pocket of Hsp90. Mahanine also reduced in vitro migration and tube formation in cancer cells. Further, it inhibited orthotopic pancreatic tumor growth in nude mice. Taken together, these results provide evidence for mahanine‐induced ROS‐mediated destabilization of Hsp90 chaperone activity resulting in Hsp90‐Cdc37 disruption leading to apoptosis, suggesting its potential as a specific target in pancreatic cancer.