Lymphatic Metastasis Of Pancreatic Cancer Research Articles

Up-regulation of sialyltransferases increases lymphatic metastasis in pancreatic cancer via the integrin-mediated pathway

Up-regulation of sialyltransferases increases lymphatic metastasis in pancreatic cancer via the integrin-mediated pathway

Src/STAT3 signaling pathways are involved in KAI1-induced downregulation of VEGF-C expression in pancreatic cancer.

A previous study by our group demonstrated that overexpression of KAI1 was associated with lymphatic metastasis in pancreatic cancer. The present study further investigated the signaling pathways involved in KAI1‑induced downregulation of vascular endothelial growth factorC (VEGF‑C) and lymphatic metastasis in pancreatic cancer. Immunohistochemistry was performed to examine KAI1 and VEGF‑C expression in 28 surgically resected pancreatic cancer tissues. MIA PaCa‑2 and PCAN1 pancreatic cancer cell lines were transfected with KAI1 overexpression vector. VEGF‑C expression as well as phosphorylation of Src and signal transducer and activator of transcription (STAT)3 were assessed by western blot analysis. Furthermore, the signal transduction inhibitors PP2 and AG490 were used to block the Src and STAT3 signaling pathways, respectively. KAI1 was negatively correlated with VEGF‑C expression in pancreatic tumor samples. In MIA PaCa‑2 cells, VEGF‑C expression was more significantly inhibited by restoration of KAI1 than that in PCAN1 cells. In addition, Src and STAT3 phosphorylation was decreased by KAI1 in MIA PaCa‑2 cells. Of note, pre‑treatment with PP2 efficiently reversed the KAI1-induced enhancement of Src and STAT3 phosphorylation as well as VEGF‑C expression. Pre‑treatment with AG490 efficiently reversed the KAI1-induced enhancement of STAT3 phosphorylation and VEGF‑C expression, but had no effect on the upregulation of Src phosphorylation. The present study identified the involvement of Src/STAT3 signaling pathways in KAI1‑induced downregulation of VEGF‑C expression and suggested the implication of these pathways in lymphatic metastasis of pancreatic cancer.

KAI1 inhibits lymphangiogenesis and lymphatic metastasis of pancreatic cancer in vivo

Several studies have shown that KAI1 inhibits tumor metastasis, but its mechanism is not clear. The present study aimed to determine the role of KAI1 in lymphatic metastasis, specifically in pancreatic cancer. The KAI1 gene was transfected into the pancreatic cancer cell line MIA PaCa-2 and PANC-1 by using liposomes and selected by G418, and the protein was measured by Western blotting. After successful infection, the cell growth curve was studied by MTT, vascular endothelial growth factor C (VEGF-C) secretion by pancreatic cancer cell were measured by ELISA. The KAI1 and pCMV transfected MIA PaCa-2 cells were renamed as MIA PaCa-2-K and MIA PaCa-2-p. These two kinds of cells were injected into the subcuticular layer of nude mice; both tumor growth and metastasis through the lymphatic nodes were assessed. Lymphangiogenesis in tumors was measured by immunohistochemistry. The VEGF-C secretion was significantly reduced in MIA PaCa-2 cells compared with PANC-1 cells after being transfected with the KAI1 gene. The growth rate of subcutaneous tumors was similar after the injection of MIA PaCa-2-K, MIA PaCa-2, and MIA PaCa-2-p. MIA PaCa-2-K tumors showed slower lymphangiogenesis and lymph node metastasis compared with MIA PaCa-2 and MIA PaCa-2-p tumors. The overexpression of KAI1 inhibits the lymphangiogenesis and lymph node metastasis of MIA PaCa-2 pancreatic tumors.

Molecular mechanism underlying lymphatic metastasis in pancreatic cancer.

As the most challenging human malignancies, pancreatic cancer is characterized by its insidious symptoms, low rate of surgical resection, high risk of local invasion, metastasis and recurrence, and overall dismal prognosis. Lymphatic metastasis, above all, is recognized as an early adverse event in progression of pancreatic cancer and has been described to be an independent poor prognostic factor. It should be noted that the occurrence of lymphatic metastasis is not a casual or stochastic but an ineluctable and designed event. Increasing evidences suggest that metastasis-initiating cells (MICs) and the microenvironments may act as a double-reed style in this crime. However, the exact mechanisms on how they function synergistically for this dismal clinical course remain largely elusive. Therefore, a better understanding of its molecular and cellular mechanisms involved in pancreatic lymphatic metastasis is urgently required. In this review, we will summarize the latest advances on lymphatic metastasis in pancreatic cancer.

The microRNA-218 and ROBO-1 signaling axis correlates with the lymphatic metastasis of pancreatic cancer

Pancreatic cancer is known for its poor prognosis and early lymphatic metastasis is a notable characteristic. microRNAs (miRNAs) have been shown to be involved in the initiation and progression of pancreatic cancer. We, therefore, established a screening strategy to find miRNAs related to the lymphatic metastasis of pancreatic cancer and explored the target genes of miRNAs. miRNA array profiles were analyzed in tissue samples [pancreatic ductal adenocarcinoma (PDAC) and matched adjacent benign tissues (MAT)] and cell lines (BxPC-3-LN and BxPC-3). Combined analysis of profiling data from tissue samples and cell lines was used to identify miRNAs related to the lymphatic metastasis of pancreatic cancer. The expression levels of miRNAs were confirmed by real‑time reverse transcription PCR (RT-PCR) in tissue samples and cell lines. The correlation between miRNAs and clinicopathological characteristics was investigated. The expression features of miRNAs in pancreatic cancer, precursor lesions and metastatic lymph nodes were characterized by in situ hybridization (ISH). Predicted target genes of miRNAs were validated by RT-PCR and the protein levels of target genes were revealed by western blotting. Seventy and 63 miRNAs were differentially expressed in pancreatic cancer and BxPC-3-LN, compared to MAT and BxPC-3, respectively. Combined microarray analysis found 4 co-differentially expressed miRNAs (miRNA-663, miRNA-145, miRNA-218 and let-7) related to the lymphatic metastasis of pancreatic cancer. miRNA-218 was significantly downregulated in BxPC-3-LN (fold-change>10) and the expression levels of miRNA-218 were confirmed by RT-PCR. The group with lymph node metastasis and the elder group (age>64) showed lower expression of miRNA-218 (P=0.003 and 0.002), compared to patients without lymph nodes metastasis and patients in the younger group (age≤64), respectively. The expression of miRNA‑218 showed a decreasing trend from normal acinar/ductal epithelium, intraductal papillary mucinous neoplasm (IPMN), pancreatic cancer to metastatic lymph nodes by ISH. Among 8 predicted target genes of miRNA-218, rodent bone (ROBO-1) was confirmed to be upregulated in both mRNA and protein levels in pancreatic cancer. In conclusion, we established a screening strategy based on microarray results and found miRNA-218 to be a notable gene related to lymphatic metastasis of pancreatic cancer. Downregulation of miRNA-218 and upregulation of ROBO-1 were first demonstrated in pancreatic cancer. The miRNA-218 and ROBO-1 signaling axis may contribute to the lymphatic metastasis of pancreatic cancer.

Highly lymphatic metastatic pancreatic cancer cells possess stem cell-like properties

Cancer stem cells are thought to be the origin of tumor metastasis. However, evidence of cancer stem cells as the source of lymphatic metastasis in pancreatic cancer is not clear. In this study, we examined the stem cell-like properties of the highly lymphatic metastatic pancreatic cancer cells BxPC-3-LN. Compared with the parental BxPC-3 cells, the BxPC-3-LN cells showed stem cell-like properties, including high lymphatic metastasis potential, self-renewal ability and chemoresistance. In addition, the BxPC-3-LN cells also expressed higher levels of sonic hedgehog and migrating cancer stem cell surface markers (CD133 and CXCR4) compared to the parental BxPC-3 cells. The growth of BxPC-3-LN cells was significantly inhibited by gemcitabine combined with the sonic hedgehog inhibitor cyclopamine. The BxPC-3-LN cells expressed lower levels of let-7, miR-34, miR-107, miR-125, miR-128, miR-130, miR-132 and miR-141 than the parental BxPC-3 cells detected by microRNA PCR array, which were reported to have close relation to stem cell factors. This study provides evidence that cancer stem cells are the major sources of pancreatic cancer lymphatic metastasis, and microRNAs may regulate lymphatic metastasis in pancreatic cancer through modulating cancer stem cells.

Development of a unique mouse model for pancreatic cancer lymphatic metastasis

Lymphatic metastasis of pancreatic cancer is a predictor of poor prognosis. However, the molecular mechanisms are largely unknown, thus, making the development of appropriate cell lines and experimental models critically important for future investigations. The purpose of the present study was to establish a 'pancreatic cancer cell and mouse model with high lymphatic metastasis potential' for in-depth study of the underlying mechanisms. The BxPC-3-LN subline, derived from the BxPC-3 human pancreatic cancer cell line, was established through serial passages in nude mice via footpad injections. The subline was able to develop notable lymphatic metastases in 100% of the recipient mice 8 weeks after tumor cell implantation. Compared with the parental BxPC-3 cells, BxPC-3-LN cells were more aggressive, displaying invasive ultrastructure, increased migration and invasion ability, and chemoresistance. Metastasis-related gene alteration including upregulation of MMP14, MMP24, MIF and ADRM1, and downregulation of TGFB2 and ROBO1 were also observed in BxPC-3-LN cells by cDNA microarrays. Thus, the newly selected BxPC-3-LN subline can serve as a unique model for further study of lymphatic metastasis of pancreatic cancer.

MicroRNA-218: A potential regulator of lymphatic metastasis in pancreatic ductal adenocarcinoma.

e14651 Background: Early lymph node metastasis is one of the most important characteristic features of pancreatic ductal adenocarcinoma (PDAC) with a dismal prognosis. MicroRNAs (miRNAs) have been shown to have a role in oncogenesis, invasion, and metastasis via epigenetic posttranscriptional gene regulation. However lymphatic metastasis related miRNAs of pancreatic cancer (PC) has not been well documented. Methods: Through microarray analysis aberrantly expressing miRNAs potentially regulating lymphatic metastasis in PC were gained from PDAC tissue specimens and cell lines. Candidate miRNAs were confirmed in total 40 resected PDAC cases and cell lines by quantitative reverse transcription-PCR (qRT-PCR). In situ hybridization was performed to elucidate the expression features of miRNA-218 in PDAC, precursor lesions, metastatic lymph nodes and cell lines. Results: MiRNA-218 and other five miRNAs were found aberrantly expressing both in PDAC and BxPC-3-LN5 (cell line with high-metastasis characteristics) by microarray analysis. Down-regulated expression of miRNA-218 was validated in PDAC tissue samples (4 fold decrease) compared to adjacent benign tissue (p=0.006) and cell line BxPC-3-LN5 (3 fold decrease) compared to BxPC-3 (p=0.01). Clinicopathological data demonstrated inverse correlation of miRNA-218 expression with lymph node metastasis (p=0.045). Expression of miRNA-218 was down-regulated in IPMN and even decreased in PDAC in a grade-dependent manner. Almost negative expression of miRNA-218 was revealed in metastatic lymph nodes. Conclusions: Down-regulated expression of miRNA-218 in PDAC and its expression features in precursor lesions suggests a tumor suppressor action of miRNA-218 in PDAC. Decreased expression of miRNA-218 may be associated with lymphatic metastasis of PDAC and interpret the high-metastasis ability of BxPC-3-LN5 cell line.

Angiopoietin‐2 drives lymphatic metastasis of pancreatic cancer

Lymphatic metastasis constitutes a critical route of disease dissemination, which limits the prognosis of patients with pancreatic ductal adenocarcinoma (PDAC). As lymphangiogenesis has been implicated in stimulation of lymphatic metastasis by vascular endothelial growth factor-C (VEGF-C) and VEGF-D, we studied the effect of the angioregulatory growth factor angiopoietin-2 (Ang-2) on PDAC progression. Ang-2 was found to be expressed in transformed cells of human PDAC specimens, with corresponding Tie-2 receptors present on blood and lymphatic endothelium. In vitro in PDAC cells, Ang-2 was subject to autocrine/paracrine TGF-β stimulation (2-fold induction, P=0.0106) acting on the -61- to +476-bp element of the human Ang-2 promoter. In turn, Ang-2 regulated the expression of genes involved in cell motility and tumor suppression. Orthotopic PDAC xenografts with forced expression of Ang-2, but not Ang-1, displayed increased blood and lymphatic vessel density, and an enhanced rate of lymphatic metastasis (6.7- to 9.1-fold, P<0.01), which was prevented by sequestration of Ang-2 via coexpression of soluble Tie-2. Notably, elevated circulating Ang-2 in patients with PDAC correlated with the extent of lymphatic metastasis. Furthermore, median survival was reduced from 28.4 to 7.7 mo in patients with circulating Ang-2 ≥ 75th percentile (P=0.0005). These findings indicate that Ang-2 participates in the control of lymphatic metastasis, constitutes a noninvasive prognostic biomarker, and may provide an accessible therapeutic target in PDAC.

Farnesoid X receptor, overexpressed in pancreatic cancer with lymph node metastasis promotes cell migration and invasion

Background:Lymph node metastasis is one of the most important adverse prognostic factors for pancreatic cancer. The aim of this study was to identify novel lymphatic metastasis-associated markers and therapeutic targets for pancreatic cancer.Methods:DNA microarray study was carried out to identify genes differentially expressed between 17 pancreatic cancer tissues with lymph node metastasis and 17 pancreatic cancer tissues without lymph node metastasis. The microarray results were validated by real-time PCR. Immunohistochemistry and western blotting were used to examine the expression of farnesoid X receptor (FXR). The function of FXR was studied by small interfering RNA and treatment with FXR antagonist guggulsterone and FXR agonist GW4064.Results:Farnesoid X receptor overexpression in pancreatic cancer tissues with lymph node metastasis is associated with poor patient survival. Small interfering RNA-mediated downregulation of FXR and guggulsterone-mediated FXR inhibition resulted in a marked reduction in cell migration and invasion. In addition, downregulation of FXR reduced NF-κB activation and conditioned medium from FXR siRNA-transfected cells showed reduced VEGF levels. Moreover, GW4064-mediated FXR activation increased cell migration and invasion.Conclusions:These findings indicated that FXR overexpression plays an important role in lymphatic metastasis of pancreatic cancer and that downregulation of FXR is an effective approach for inhibition of pancreatic tumour progression.

Expression of cyclooxygenase-2, vascular endothelial growth factor-c and CD44v6 proteins and its relation with lymphatic metastasis of pancreatic cancer

Objective To investigate the expression of cyclooxygenase-2 (COX-2), vascular en-dothelial growth factor-c (VEGF-C) , CD44v6 in pancreatic cancer tissue and its relation with the ctinico-pathologic features. Methods The expression of COX-2, VEGF-C and CD44v6 proteins in 48 cases of pancreatic cancer was detected by immnohistochemical staining. Results The positive rate of COX-2, VEGF-C and CD44v6 protein in pancreatic cancer tissue was 79.2% ,58.3%, and 62.5%, respectively, which was higher than in adjacent tissues (0/14) ( P 0.05 ), but that of VEGF-C and CD44V6 remarkably with lymph node metastasis ( P 0.05 ). Conclusion The expres-sion of COX-2, VEGF-C and CD44v6 in pancreatic cancer tissue might play a role in lymphatic metastasis via lymphangiogenesis. Key words: Pancreatic neoplasm; Cyclooxygenase-2; Vascular endothelial growth factor

Significance of VEGF-C and VEGF-D in lymphatic metastasis of pancreatic cancer

Objective To analyze the intratumoral and peritumoral microvessel density (MVD) and microlymphatic vessel density (MLVD) in pancreatic cancer and record the expression of vascular endothelial growth factor(VEGF)-C and VEGF-D. And to explore the significance of VEGF-C and VEGF-D during the lymphatic metastasis and development of pancreatic cancer. Methods The expression of VEGF-C and VEGF-D, VEGF-R3, CD34 were assayed by immunohistochemical staining in 30 cases of pancreatic cancer tissues. Results The positive rates of VEGF-C and VEGF-D protein in the central portion of tumors (30% and 16.7%) were significantly lower than those in the marginal portion (73.3% and 56.7%), P <0.01. The group with high expression of VEGF-C and VEGF-D in the marginal portion had significantly higher incidences of lymph node metastasis, lymphatic invasion and venous invasion( P <0. 01 ). MLVD in both of the VEGF-C and VEGF-D positive groups was higher than that in the negative groups( P <0. 01 ), and the lymph node me-tastasis increased. MVD in VEGF-C positive group was significantly higher than that in the negative group. MVD had no significant difference between VEGF-D positive and negative group ( P =0. 07). Conclusions The expression of VEGF-C and VEGF-D in the marginal portion of tumor is significantly correlated with lym-phatic metastasis in pancreatic cancer patients, and may induce lymphangiogenesis. VEGF-C may play an im-portant role in the regulation of angiogenesis and lymphangiogenesis in pancreatic cancer, and VEGF- D maybe only participate in the regulation of lymphangiogenesis. Key words: Vascular endothelial growth factors; Pancreatic neoplasms; Lymphangiogenesis; Lym-phatic metastasis

Relationship of expressions of vascular endothelial growth factor and number of tumour-associated macrophages with lymphatic metastasis in pancreatic cancer

目的 研究胰腺癌组织中血管内皮生长因子(vascular endothelial growth factor,VEGF)表达和肿瘤相关巨噬细胞(tumour-associated macrophages,TAMs)计数与淋巴结转移的关系.方法 采用免疫组织化学染色,在光镜下对VEGF阳性细胞评分及TAMs计数进行分析.结果 胰腺癌组织中VEGF表达阳性率明显高于正常组织(P＜0.01);淋巴结转移组VEGF的表达和TAMs计数较未转移组明显增高(P＜0.05);VEGF的表达与胰腺癌临床病理分期无明显关系(P＞0.05);TAMs计数与胰腺癌临床病理分期有明显关系(P＜0.01);VEGF的表达与TAMs计数呈正相关(r=0.663,P＜0.01).结论 VEGF和TAMs的表达均与胰腺癌淋巴结转移有密切关系。

Analysis of genes associated with lymphatic metastasis in pancreatic carcinoma using cDNA microarray

Objective: To identify new markers for prediction of lymph node metastasis. Methods: cDNA probes were prepared by labeling mRNA from samples of four pancreatic carcinoma tissues with Cy5-dUTP and mRNA from adjacent normal tissues with Cy3-dUTP respectively through reverse transcription. The mixed probes of each sample were then hybridized with 4,096 cDNA arrays (4,000 unique human cDNA sequences), and the fluorescent signals were scanned by ScanArray 3000 scanner (General Scanning, Inc.). The values of Cy5-dUTP and Cy3-dUTP on each spot were analyzed and calculated by ImaGene 3.0 software (BioDiscovery, Inc.). Genes that differentially expresses in each cancerous tissue were sought out according to the standard that the absolute value of natural logarithm of the ratio of Cy5 to Cy3 is greater than 0.69, i. e., more than 2 times change of gene expression, and the signal value of either Cy3 and Cy5 need to be greater than 600. Then, the genes differently expressed in cancer with and without lymphatic metastasis were screened out for further analysis. Results: Among 2 samples with lymphatic metastasis and 2 samples without metastasis, 56 genes, which accounted for 1.40% of genes on the microarray slides, exhibited differentially expression in cancerous tissues with lymphatic metastasis. There were 32 over-expressed genes including 11 having been registered in Genebank, and 24 under-expressed genes including 3 in Genebank. Conclusion: Microarray analysis may provide invaluable information to identify specific gene expression profile of lymphatic metastasis in pancreatic cancer.