[Show abstract][Hide abstract] ABSTRACT: BackgroundMUC4 plays important roles in the malignant progression of human pancreatic cancer. But the huge length of MUC4 gene fragment restricts its functional and mechanism research. As one of its splice variants, MUC4/Y with coding sequence is most similar to that of the full-length MUC4 (FL-MUC4), together with alternative splicing of the MUC4 transcript has been observed in pancreatic carcinomas but not in normal pancreas. So we speculated that MUC4/Y might be involved in malignant progression similarly to FL-MUC4, and as a research model of MUC4 in pancreatic cancer. The conjecture was confirmed in the present study.MethodsMUC4/Y expression was detected by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) using gene-specific probe in the clinic samples. The effects of MUC4/Y were observed by serial in vitro and in vivo experiments based on stable over-expressed cell model. The underlying mechanisms were investigated by sequence-based transcriptome analysis and verified by qRT-PCR, Western blot and enzyme-linked immunosorbent assays.ResultsThe detection of clinical samples indicates that MUC4/Y is significantly positive-correlated with tumor invasion and distant metastases. Based on stable forced-expressed pancreatic cancer PANC-1 cell model, functional studies show that MUC4/Y enhances malignant activity in vitro and in vivo, including proliferation under low-nutritional-pressure, resistance to apoptosis, motility, invasiveness, angiogenesis, and distant metastasis. Mechanism studies indicate the novel finding that MUC4/Y triggers malignancy-related positive feedback loops for concomitantly up-regulating the expression of survival factors to resist adverse microenvironment and increasing the expression of an array of cytokines and adhesion molecules to affect the tumor milieu.Conclusions
In light of the enormity of the potential regulatory circuitry in cancer afforded by MUC4 and/or MUC4/Y, repressing MUC4 transcription, inhibiting post-transcriptional regulation, including alternative splicing, or blocking various pathways simultaneously may be helpful for controlling malignant progression. MUC4/Y- expression model is proven to a valuable tool for the further dissection of MUC4-mediated functions and mechanisms.
Journal of Translational Medicine 11/2014; 12(1):309. · 3.99 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Natural killer (NK) cells play a key role in non-specific immune response in different cancers, including pancreatic cancer. However the anti-tumor effect of NK cells decreases during pancreatic cancer progression. The regulatory pathways by which NK cells facilitate tumor immune escape are unclear, therefore our purpose was to investigate the roles of the contributory factors.
BMC Cancer 10/2014; 14(1):738. · 3.32 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Background
Hepatocellular carcinoma (HCC) typically relies on tumor transformation and angiogenesis for its malignant behavior, including growth and metastasis. Previously, we reported that Vasohibin2 (VASH2) is preferentially expressed in hepatocellular carcinoma (HCC) tumor tissues and promotes angiogenesis. Here, we further investigated the role of VASH2 in HCC tumor progression.ResultsBioinformatics analyses and luciferase reporter gene assays confirmed the post-transcriptional regulation of VASH2 by miR-200a/b/c. We then used HepG2 and Hep3B cells, two representative hepatic cancer cell lines, to examine the role of VASH2 in tumors. VASH2 knockdown in HepG2 cells inhibited epithelial-mesenchymal transition (EMT), but VASH2 overexpression in Hep3B cells promoted EMT. Western blot analyses showed that VASH2 promoted EMT through the ZEB1/2 pathway.ConclusionVASH2 promoted invasion, reduced apoptosis and increased the proportion of stem cells in vitro and in vivo. These results indicated that VASH2 expression in HCC cells promotes the malignant transformation of tumors by inducing EMT.
Cell Communication and Signaling 10/2014; 12(1):62. · 4.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Vasohibin‑2 (VASH2) is an angiogenic factor, and has been previously reported to be a cancer‑related gene, with cytoplasmic and karyotypic forms. In the current study VASH2 expression in human breast cancer tissue and adjacent non‑cancerous tissue was investigated with immunohistochemistry. MCF‑7 and BT474 human breast cancer cells were transfected with lentiviral constructs to generate in vitro VASH2 overexpression and knockdown models. In addition, BALB/cA nude mice were inoculated subcutaneously with transfected cells to generate in vivo models of VASH2 overexpression and knockdown. The effect of VASH2 on cell proliferation was investigated using a bromodeoxyuridine assay in vitro and immunohistochemistry of Ki67 in xenograft tumors. Growth factors were investigated using a human growth factor array, and certain factors were further confirmed by an immunoblot. The results indicated that the expression level of cytoplasmic VASH2 was higher in breast cancer tissues with a Ki67 (a proliferation marker) level of ≥14%, compared with tissues with a Ki67 level of <14%. VASH2 induced proliferation in vitro and in vivo. Four growth factors activated by VASH2 were identified as follows: Fibroblast growth factor 2 (FGF2), growth/differentiation factor‑15 (GDF15), insulin‑like growth factor‑binding protein (IGFBP)3 and IGFBP6. FGF2 and GDF15 may contribute to VASH2‑induced proliferation. The current study identified a novel role for VASH2 in human breast cancer, and this knowledge suggests that VASH2 may be a novel target in breast cancer treatment.
Molecular Medicine Reports 06/2014; · 1.48 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Increasing evidence indicates an important role of transcription factor Yin Yang-1 (YY1) in human tumorigenesis. However, its function in cancer remains controversial and the relevance of YY1 to pancreatic ductal adenocarcinoma (PDAC) remains to be clarified.
Molecular Cancer 05/2014; 13(1):130. · 5.40 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Pancreatic ductal adenocarcinoma (PDAC) is a highly invasive cancer with a poor prognosis. Although microRNA (miRNA) transcripts have a crucial role in carcinogenesis and development, little information is known regarding the aberrant DNA methylation of miRNAs in PDAC. Using methylated DNA immunoprecipitation-chip analysis, we found that miR-615-5p was hypermethylated in its putative promoter region, which silenced its expression in PDAC cell lines. In addition, the overexpression of miR-615-5p in pancreatic cancer cells suppressed cell proliferation, migration and invasion. Insulin-like growth factor 2 (IGF2) is an imprinted gene, and its abnormal expression contributes to tumor growth. Here, we identified IGF2 as a target of miR-615-5p using a luciferase reporter assay. IGF2 upregulation in PDAC tissues was not correlated with a loss of imprinting but was inversely correlated with miR-615-5p downregulation. In addition, miR-615-5p suppressed pancreatic cancer cell proliferation, migration and invasion by directly targeting IGF2, and this effect could be reversed by co-transfection with IGF2. Furthermore, the stable overexpression of miR-615-5p inhibited tumor growth in vivo and was correlated with IGF2 expression. Using RNA sequencing, we further identified miR-615-5p as potentially targeting other genes, such as the proto-oncogene JUNB, and interfering with the insulin signaling pathway. Taken together, our results demonstrate that miR-615-5p was abnormally downregulated in PDAC cells due to promoter hypermethylation, which limited its inhibition of IGF2 and other target genes, thereby contributing to tumor growth, invasion and migration. These data demonstrate a novel and important role of miR-615-5p as a tumor suppressor in PDAC.Oncogene advance online publication, 28 April 2014; doi:10.1038/onc.2014.101.
[Show abstract][Hide abstract] ABSTRACT: Hepatocellular carcinoma (HCC) is a prevalent problem worldwide. Chemotherapy, especially cisplatin (CDDP)-based systemic chemotherapy, is the best option for advanced liver cancer. However, CDDP resistance is becoming common and hindering the clinical application of CDDP. Meanwhile, no consensus has been reached regarding the chemotherapeutic use of vasohibin 2 (VASH2), which promotes the angiogenesis and proliferation of cancer cells. In this work, a tissue microarray was used to observe VASH2 and its possible role in cancer treatment. Results showed that VASH2 was highly expressed in HCC tissues and was significantly correlated with cancer differentiation. To further investigate the efficacy and mechanism of the combination of VASH2 with anti-cancer drugs in liver cancer cells, we stably built VASH2 overexpression and knockdown cell lines. We found that VASH2 can influence the CDDP sensitivity and that the cell overexpression of VASH2 had a higher cell viability and lower apoptosis rate after CDDP exposure. We also observed that VASH2 overexpression downregulated wild-type p53, as well as suppressed the expression of the pro-apoptotic protein BCL2-associated X protein (Bax) and cleaved caspase-3 (CC-3) after treatment by CDDP. Conversely, the knockdown of VASH2 significantly inhibited these effects. In an in vivo chemosensitivity study, nude mice were subcutaneously injected with tumor cells and received CDDP treatment through intraperitoneal administration every 3 days. We found that VASH2 knockdown markedly limited the tumor growth and enhanced the CDDP toxicity and apoptosis of tumor cells. Western blot analysis revealed that tumor cells with downregulated VASH2 had a higher expression of wild-type p53, Bax, and CC-3 than control cells. Overall, our results indicated the novel roles of VASH2 in the chemoresistance of hepatocarcinoma cells to CDDP and suggested that VASH2 may be a promising anticancer target.
PLoS ONE 03/2014; 9(3):e90358. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Galectin-1, a member of carbohydrate-binding proteins with a polyvalent function on tumor progression, was found strongly expressed in pancreatic satellite cells (PSCs), which partner in crime with cancer cells and promote the development of pancreatic ductal adenocarcinoma (PDAC). We evaluated the effects of PSCs derived Galectin-1 on the progression of PDAC, as well as the tumor establishment and development in mouse xenografts.
The relationship between immunohistochemistry staining intensity of Galectin-1 and clinicopathologic variables were assessed in 66 PDAC tissues, 18 chronic pancreatitis tissues and 10 normal controls. The roles of PSCs isolated from PDAC and normal pancreas on the proliferative activity, MMP2 and MMP9 expression, and the invasion of CFPAC-1 in the co-cultured system, as well as on the tumor establishment and development in mouse xenografts by mixed implanting with CFPAC-1 subcutaneously were evaluated.
Galectin-1 expression was gradually increased from normal pancreas (negative), chronic pancreatitis (weak) to PDAC (strong), in which Galectin-1 expression was also increased from well, moderately to poorly differentiated PDAC. Galectin-1 staining intensity of pancreatic cancer tissue was associated with increase in tumor size, lymph node metastasis, perineural invasion and differentiation and UICC stage, and served as the independent prognostic indicator of poor survival of pancreatic cancer. In vitro and in vivo experiments indicated that TGF-β1 upregulated Galectin-1 expression in PSCs, which could further promotes the proliferative activity, MMP2 and MMP9 expression, and invasion of pancreatic cancer cells, as well as the tumor establishment and growth.
Galectin-1 expression in stromal cells of pancreatic cancer suggests that this protein plays a role in the promotion of cancer cells invasion and metastasis and provides a therapeutic target for the treatment of pancreatic cancer.
PLoS ONE 03/2014; 9(3):e90476. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Tumor-associated MUC4 mucin has considerable potential as an immunotherapy target for pancreatic cancer. In previous studies, we developed dendritic cell (DC) vaccines which elicited MUC4 antigen-specific cytotoxic T lymphocyte (MS-CTL) response against tumor cells in vitro. Due to the observation that MS-CTL apoptotic rate increased significantly when co-cultured with MUC4+ tumor cells compared with T2 cells, we investigated whether high expression levels of MUC4 in pancreatic cancer cells would have an effect on the significant increase of apoptosis rate of MS-CTLs. First, the adverse influence of regulatory T cells (Tregs) was eliminated by CD8+ T lymphocyte sorting before the induction of MS-CTLs. Then, we constructed clonal MUC4-knockdown HPAC pancreatic cancer sublines with different MUC4 expression for co-incubation system. By utilizing appropriate control to rule out the possible apoptosis-induced pathway of intrinsic activated cell-autonomous death (ACAD) and analogous antigen-dependent apoptosis of CTL (ADAC) in our study system, further analysis of the effect of MUC4 membrane-expression, supernatants and blockade of CTL surface Fas receptor on MS-CTL apoptosis was carried out. The results demonstrated that the level of MUC4 membrane expression strongly positively correlated with MS-CTL apoptosis and the influence of supernatants and Fas-blockade did not significantly correlate with MS-CTL apoptosis. This evidence suggested that there may be a novel counterattack pathway of pancreatic cancer cells, which is a MUC4-mediated, cell contact-dependent and Fas-independent process, to induce CTL apoptosis. Therefore, further exploration and understanding of the potential counterattack mechanisms is beneficial to enhance the efficacy of MUC4 specific tumor vaccines.
[Show abstract][Hide abstract] ABSTRACT: MiR-106b-25 cluster, hosted in intron 13 of MCM7, may play integral roles in diverse processes including immune response, tumorigenesis and progression. A single nucleotide polymorphism (SNP), rs999885, is located in the promoter region of MCM7. Our previous study showed that the A to G base change of rs999885 may provide an increased risk for HCC in HBV persistent carriers by altering the expression of the miR-106b-25 cluster. However, it is unknown whether rs999885 is associated with prognosis of intermediate or advanced HBV-related hepatocellular carcinoma (HCC) patients.
The SNP, rs999885, was genotyped by using the TaqMan allelic discrimination Assay in 414 intermediate or advanced HCC patients. Log-rank test and Cox proportional hazard models were used for survival analysis.
The variant genotypes of rs999885 were associated with a significantly decreased risk of death for intermediate or advanced HCC [additive model: adjusted hazard ratio (HR) = 0.76,95% confidence intervals (CI) = 0.59-0.97]. Further stepwise regression analysis suggested that rs999885 was an independently protective factor for the prognosis of HCC in the final model (additive model: adjusted HR = 0.72, 95% CI = 0.56-0.91, P = 0.007).
These findings indicate that the A to G base change of rs999885 may provide a protective effect on the prognosis of intermediate or advanced HCC in Chinese.
PLoS ONE 01/2014; 9(1):e85394. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Expression of the transmembrane-4-L-six-family-1 (TM4SF1) is high in human pancreatic cancer cells, but the underlying mechanism remains unclear. In this study, we aimed to identify and characterize microRNAs that regulate TM4SF1 expression in PC cells. Western blot analysis and quantitative polymerase chain reaction were used to detect TM4SF1 and hsa-miR-141 levels in four PC cell lines. SW1990 and BxPc-3 cells were transfected with the inhibitor miR-141, the inhibitor negative control, the miR-141 mimic and the mimic negative control; and cell invasion, migration, proliferation, cell cycle progression and apoptosis were detected by Transwell, MTT and flow cytometry assays, respectively. The miR-141 levels negatively correlated with the TM4SF1 protein levels in PC cells. The TM4SF1 protein levels were lower in the 141M group but higher in the 141I group, although the TM4SF1 mRNA levels had no significant changes, compared to the negative controls. Luciferase assays demonstrated that hsa-miR-141 directly targeted the 3'-untranslated region of the TM4SF1 gene. In addition, miR-141 downregulated TM4SF1 expression to inhibit invasion and migration of PC cells but had no effects on cell proliferation, cell cycle progression or apoptosis. TM4SF1 is a direct target of miR-141. Our findings that TM4SF1 expression was inhibited by miR-141 provide new insights into the oncogenic mechanism of TM4SF1 and suggest that miR-141 represents a novel molecular target for PC therapy.
International Journal of Oncology 11/2013; · 2.77 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The tumor microenvironment plays an important role in the progression of cancer. This study focused on carcinoma-associated fibroblasts (CAFs) and stromal-epithelial interaction between CAFs and epithelial ovarian carcinoma (EOC) cells. We isolated and established primary cultures of CAFs and co-cultured CAFs and EOC cells in vitro. The co-culture conditioned medium (CC-CM) was harvested and its influence on EOC cells was examined. Cytokine, chemokine, and growth factor levels were screened using a biotin label-based human antibody array system. We found that the stromal-epithelial crosstalk provided a suitable microenvironment for the progression of ovarian cancer cells in vitro.
Cancer Investigation 10/2013; · 2.24 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Digestive malignancies, especially pancreatic cancer (PC), gastric cancer (GC), and colorectal cancer (CRC), still occur at persistently high rates, and disease progression in these cancers has been associated with tumor immunosurveillance escape. Natural killer (NK) cell dysfunction may be responsible for this phenomenon, however, the exact relationship between tumor immunosurveillance escape in digestive malignancies and NK cell dysfunction remains unclear.
Percentage of the surface receptors NKG2A, KIR3DL1, NKG2D, NKp30, NKp44, NKp46, and DNAM-1, as well as the cytotoxic granules perforin and granzyme B positive NK cells were determined in patients with pancreatic cancer (n = 31), gastric cancer (n = 31), and CRC (n = 32) prior to surgery and healthy controls (n = 31) by multicolor flow cytometry. Independent t-tests or Mann-Whitney U-tests were used to compare the differences between the patient and healthy control groups, as well as the differences between patients with different pathologic features of cancer.
Percentage of NKG2D, NKp30, NKp46, and perforin positive NK cells was significantly down-regulated in patients with PC compared to healthy controls, as well as GC and CRC; reduced levels of these molecules was associated with indicators of disease progression in each malignancy (such as histological grade, depth of invasion, lymph node metastasis). On the contrary, percentage of KIR3DL1 positive NK cells was significantly increased in patients with PC, as well as GC and CRC, but was not associated with any indicators of disease progression.
Altered percentage of surface receptors and cytotoxic granules positive NK cells may play a vital role in tumor immunosurveillance escape by inducing NK cell dysfunction in patients with PC, GC, and CRC.
Journal of Translational Medicine 10/2013; 11(1):262. · 3.99 Impact Factor
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[Show abstract][Hide abstract] ABSTRACT: This review focuses on the state-of-the-art of CXCL12/CXCR4 signaling axis in pancreatic cancer and its role in tumor progression.
Relevant articles published in English were identified by searching in Pubmed from 1997 to 2013, with keywords "CXCL12", "CXCR4" and "pancreatic cancer". Important references from selected articles were also retrieved.
Articles about CXCL12/CXCR4 signaling axis in pancreatic cancer and relevant mechanisms were selected.
Pancreatic cancer has been one of the most lethal human malignancies, with median survival less than one year and overall 5-year survival only 6%. Tumor cells from pancreatic cancer express high level of CXCR4. CXCL12, the ligand for CXCR4, is extensively secreted by neighboring stromal cells and other distant organs. CXCL12 primarily binds to CXCR4, induces intracellular signaling through several divergent pathways, which are involved in progression and metastasis of pancreatic cancer.
CXCL12/CXCR4 signaling axis may play an important role in the communication between pancreatic cancer cells and their microenvironment, which may have effect on tumor proliferation, invasion, angiogenesis, metastasis and chemoresistance. CXCL12/CXCR4 signaling axis may serves as a novel therapeutic target for pancreatic cancer.
Chinese medical journal 09/2013; 126(17):3371-4. · 1.02 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Pancreatic cancer is a disease with an extremely poor prognosis. The acquisition of invasion properties in pancreatic cancer is accompanied by the process of epithelial-mesenchymal transition (EMT). Carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) is emerging as an important determinant of the malignant phenotype in a range of cancers, including pancreatic cancer. Therefore, the aim of this study was to evaluate the potential involvement of CEACAM6 in the invasion and metastasis of pancreatic cancer cells via EMT regulation. The results of our study showed a positive association between CEACAM6 expression and poor prognosis of pancreatic cancer, differentiation and lymph node metastasis. Elevated levels of CEACAM6 in pancreatic cancer cells promoted EMT, migration and invasion in vitro and metastasis in animal models, whereas shRNA-mediated CEACAM6 knockdown had the opposite effect. Furthermore, we demonstrated that miR-29a/b/c specific for CEACAM6 could regulate its expression at the post-transcriptional level. Collectively, our findings identified CEACAM6, which is regulated by miR-29a/b/c, as an important positive regulator of EMT in pancreatic cancer offering an explanation for how elevated levels of CEACAM6 are likely to contribute to the highly metastatic phenotype of pancreatic cancer.
International Journal of Oncology 09/2013; 43(3):877-85. · 2.77 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The human mucin 4 (MUC4) is aberrantly expressed in pancreatic adenocarcinoma and tumor cell lines, while remaining undetectable in normal pancreas, indicating its important role in pancreatic cancer development. Although its transcriptional regulation has been investigated in considerable detail, some important elements remain unknown. The aim of the present study was to demonstrate the existence of a novel inhibitory element in the MUC4 promoter and characterize some of its binding proteins. By luciferase reporter assay, we located the inhibitory element between nucleotides -2530 and -2521 in the MUC4 promoter using a series of deletion and mutant reporter constructs. Electrophoretic mobility shift assay (EMSA) with Bxpc-3 cell nuclear extracts revealed that one protein or protein complex bind to this element. The proteins binding to this element were purified and identified as Yin Yang 1 (YY1) by mass spectrometry. Supershift assay and chromatin immunoprecipitation (ChIP) assay confirmed that YY1 binds to this element in vitro and in vivo. Moreover, transient YY1 overexpression significantly inhibited MUC4 promoter activity and endogenous MUC4 protein expression. In conclusion, we reported here a novel inhibitory element in the human MUC4 promoter. This provides additional data on MUC4 gene regulation and indicates that YY1 may be a potential target for abnormal MUC4 expression.
[Show abstract][Hide abstract] ABSTRACT: Vasohibin-2 was recently identified as an important pro-angiogenesis factor in solid tumor and intracellular localization of its variants is important for elucidating the downstream mechanism(s) of its effects. Currently there are no reported antibodies affordable for intracellular localization. The aim of this study was to generate and characterize polyclonal antibodies against Vasohibin-2 and to determine the intracellular localization of Vasohibin-2. In this study, two polypeptides were synthesized and one prokaryotic Vasohibin-2 recombinant protein was custom-made. New Zealand rabbits were immunized with the polypeptide mixture and prokaryotic recombinant protein, respectively. The purified antibodies from the antiserum were validated by ELISA, western blotting (WB), immunofluorescence (IF), immunohistochemistry (IHC) and immunoprecipitation (IP). In order to determine intracellular localization, the cytoplasmic and nuclear proteins of the human liver cancer cell line HepG2 were isolated for the detection of Vasohibin-2 by western blotting. Vasohibin-2 cDNA, coding for 311 and 355 amino acid residues, fused with or without a DDK/V5 tag at the c-terminus, respectively, was cloned into the Lv-CMV-EGFP vector. Lentiviruses were successfully packaged. Vasohibin-2-overexpressing HepG2-VASH2 (355 amino acid residues) and HepG2-VASH2-V5 (311 amino acid residues fused with V5 tag at the c-terminus) human liver cancer cell lines were established. Approximately 1-2x106 HepG2, HepG2-VASH2 and HepG2-VASH2-V5 cells were injected subcutaneously into the flanks of BALB/c nude mice. Xenograft tumors were harvested for immunohistochemistry. HepG2 cells were transiently transfected with the Lv-CMV-EGFP vectors containing Vasohibin-2 cDNA (coding for 311/355 amino acid residues with a DDK tag at the c-terminal), followed by anti-DDK immunofluorescence. The antibodies obtained were able to detect human VASH2 successfully as applied in western blotting, IF, IHC and IP. Results from IF, IHC and WB (post cytoplasmic/nuclear protein isolation) showed a quite different intracellular localization of VASH2 protein. The VASH2 (with 355 amino acid residues) was located in the cytoplasm while VASH2 (with 311 amino acid residues) was located in the nucleus. The former was found to be a relatively low abundance protein. We successfully generated three rabbit anti-human Vasohibin-2 polyclonal antibodies which can be used for western blotting, IF, IP and IHC. These antibodies will provide a convenient tool for further studies on Vasohibin-2. This is the first study to report differences in the intracellular localization of the VASH2 protein and, hence, a new research direction on the study of VASH2.
International Journal of Oncology 04/2013; · 2.77 Impact Factor