Lentivirus-mediated overexpression of TGF-β inducible early gene 1 inhibits SW1990 pancreatic cancer cell growth

TIEG1 (TGF-β inducible early gene 1) plays a significant role in regulating cell proliferation and apoptosis in various cell types. Previous studies have shown a close relationship between the expression level of TIEG1 and various cancers, including breast, prostate, colorectal and pancreatic cancer. In this study, we up-regulated the gene expression of TIEG1 in SW1990 pancreatic cancer cell line by a lentivirus transfection system and investigated its potential as a therapeutic target for pancreatic cancer. The results showed that lentivirus-mediated overexpression of TIEG1 gene inhibited human pancreatic cancer SW1990 cell proliferation and caused the cell cycle arrest at the G1-phase in vitro. SW1990 cells transduced with lenti-TIEG1 showed significant inhibition of colony formation and cancer cell growth in 3-D culture model. Moreover, overexpression of TIEG1 gene significantly slowed the growth of SW1990 xenografts in nude mice. Taken together, these data provided evidence that overexpression of TIEG1 gene by a lentivirus transfection system led to suppressed human pancreatic cancer cell growth and might therefore be a feasible approach in the clinical management of pancreatic cancer.     

Reduction of c-myc expression by an antisense approach under Cre/loxP switching induces apoptosis in human liver cancer cells

c-Myc has been documented to be both a positive and a negative signal for the induction of apoptosis. It is well known that overexpression of the c-myc gene induces apoptosis of normal cells, but the result of a reduction in its expression is not fully understood. We examined whether a reduction in c-myc expression would induce apoptosis in human liver cancer cells. Specifically, antisense and sense oligodeoxynucleotides (oligos) against the human c-myc mRNA were synthesized, mixed with a liposome reagent at various ratios, and were applied to the liver cancer-derived cell lines, HCC-T, HepG2, and PLC/PRF/5. To exclude effects resulting from using oligos, plasmid vectors expressing the full-length c-myc cDNA in both sense and antisense orientations under the control of the Cre/loxP system were generated. Monoclonal cell lines including these plasmid vectors were produced and Cre was supplied by adenovirus infection. Apoptosis was determined morphologically and c-Myc and Bcl-2 expression was examined by Western blotting. The antisense myc significantly inhibited the proliferation of the cells within two days, while neither the liposome reagent alone nor sense myc did so. Most of the cells were rounded up by the antisense-treatment and nuclear fragmentation and DNA ladder formation were detected after two days in antisense c-myc-treated cells. Antisense c-myc largely reduced c-Myc and partially Bcl-2 expression; overexpression of Bcl-2 partially rescued from apoptosis in HCC-T and HepG2 cells. These results suggest that the massive reduction in c-myc mRNA induces apoptosis in liver cancer cell lines and consequent decrease in Bcl-2 enhances the cell death. c-Myc reduction under the Cre/loxP switching system may be a useful tool for the clarification of c-myc-related cellular mechanisms in differentiation and proliferation.     

Jab1/Cops5 contributes to chemoresistance in breast cancer by regulating Rad51

Jab1 overexpression correlates with poor prognosis in breast cancer patients, suggestting that targeting the aberrant Jab1 signaling in breast cancer could be a promising strategy. In the current study, we investigate the hypothesis that Jab1 positively regulates the DNA repair protein Rad51 and, in turn, the cellular response of breast cancer to chemotherapy with adriamycin and cisplatin. High-throughput mRNA sequencing (RNA-Seq) data from 113 normal and 1109 tumor tissues (obtained from TCGA) were integrated to our analysis to give further support to our findings. We found that Jab1 was overexpressed in adriamycin-resistant breast cancer cell MCF-7R compared with parental MCF-7 cells, and that knockdown of Jab1 expression conferred cellular sensitivity to adriamycin and cisplatin both in vivo and in vitro. By contrast, exogenous Jab1 expression enhanced the resistance of breast cancer cells to adriamycin and cisplatin. Moreover, we discovered that Jab1 positively regulated Rad51 in p53-dependent manner and that overexpression of Rad51 conferred cellular resistance to adriamycin and cisplatin in Jab1-deficient cells. Data from TCGA further validated an correlation between Jab1 and Rad51 in breast cancer, and elevated Jab1 and Rad51 associated with poor survival in breast cancer patients. Our findings indicate that Jab1 association with Rad51 plays an important role in cellular response to chemotherapy in breast cancer.     

Beta-catenin mediates alteration in cell proliferation, motility and invasion of prostate cancer cells by differential expression of E-cadherin and protein kinase D1

We have previously demonstrated that Protein Kinase D1 (PKD1) interacts with E-cadherin and is associated with altered cell aggregation and motility in prostate cancer (PC). Because both PKD1 and E-cadherin are known to be dysregulated in PC, in this study we investigated the functional consequences of combined dysregulation of PKD1 and E-cadherin using a panel of human PC cell lines. Gain and loss of function studies were carried out by either transfecting PC cells with full-length E-cadherin and/or PKD1 cDNA or by protein silencing by siRNAs, respectively. We studied major malignant phenotypic characteristics including cell proliferation, motility, and invasion at the cellular level, which were corroborated with appropriate changes in representative molecular markers. Down regulation or ectopic expression of either E-cadherin or PKD1 significantly increased or decreased cell proliferation, motility, and invasion, respectively, and combined down regulation cumulatively influenced the effects. Loss of PKD1 or E-cadherin expression was associated with increased expression of the pro-survival molecular markers survivin, beta-catenin, cyclin-D, and c-myc, whereas overexpression of PKD1 and/or E-cadherin resulted in an increase of caspases. The inhibitory effect of PKD1 and E-cadherin on cell proliferation was rescued by coexpression with beta-catenin, suggesting that beta-catenin mediates the effect of proliferation by PKD1 and E-cadherin. This study establishes the functional significance of combined dysregulation of PKD1 and E-cadherin in PC and that their effect on cell growth is mediated by beta-catenin.     

Identification of phosphoenolpyruvate carboxykinase 1 as a potential therapeutic target for pancreatic cancer

Pancreatic cancer is the third leading cause of cancer-related mortalities and is characterized by rapid disease progression. Identification of novel therapeutic targets for this devastating disease is important. Phosphoenolpyruvate carboxykinase 1 (PCK1) is the rate-limiting enzyme of gluconeogenesis. The current study tested the expression and potential functions of PCK1 in pancreatic cancer. We show that PCK1 mRNA and protein levels are significantly elevated in human pancreatic cancer tissues and cells. In established and primary pancreatic cancer cells, PCK1 silencing (by shRNA) or CRISPR/Cas9-induced PCK1 knockout potently inhibited cell growth, proliferation, migration and invasion, and induced robust apoptosis activation. Conversely, ectopic overexpression of PCK1 in pancreatic cancer cells accelerated cell proliferation and migration. RNA-seq analyzing of differentially expressed genes (DEGs) in PCK1-silenced pancreatic cancer cells implied that DEGs were enriched in the PI3K-Akt-mTOR cascade. In pancreatic cancer cells, Akt-mTOR activation was largely inhibited by PCK1 shRNA, but was augmented after ectopic PCK1 overexpression. In vivo, the growth of PCK1 shRNA-bearing PANC-1 xenografts was largely inhibited in nude mice. Akt-mTOR activation was suppressed in PCK1 shRNA-expressing PANC-1 xenograft tissues. Collectively, PCK1 is a potential therapeutic target for pancreatic cancer.Subject terms: Pancreatic cancer, Oncogenes     

Profilin‐1 overexpression inhibits proliferation of MDA‐MB‐231 breast cancer cells partly through p27kip1 upregulation

Profilin‐1 (Pfn1), a ubiquitously expressed actin‐binding protein, has gained interest in epithelial‐derived cancer because of its downregulation in expression in various adenocarcinoma. Pfn1 overexpression impairs tumorigenic ability of human breast cancer xenografts thus suggesting that Pfn1 could be a tumor‐suppressor protein. The objective of the present study was to determine how Pfn1 overexpression affects cell‐cycle progression of breast cancer cells. We show that Pfn1 overexpression in MDA‐MB‐231 breast cancer cells causes cell‐cycle arrest in G1 phase and dramatically reduced proliferation in culture. Pfn1 overexpression results in increased protein stability of p27^kip1 (p27—a major cyclin‐dependent kinase inhibitor) and marked elevation in the overall cellular level of p27. Proliferation defect of Pfn1 overexpressers can be partly rescued by silencing p27 expression thus suggesting a critical role of p27 in Pfn1‐induced growth inhibition of MDA‐MB‐231 cells. Finally, Pfn1 overexpression was found to sensitize MDA‐MB‐231 cells to apoptosis in response to cytotoxic stimulus thus suggesting for the first time that survival of breast cancer cells can also be negatively influenced by Pfn1 upregulation. These findings may provide novel insights underlying Pfn1's tumor‐suppressive action. J. Cell. Physiol. 223:623–629, 2010. © 2010 Wiley‐Liss, Inc.     

S100A7, Jab1, and p27kip1 expression in psoriasis and S100A7 CRISPR-activated human keratinocyte cell line

Psoriasis, a chronic immune-mediated inflammatory skin disease, is characterized by dysregulated keratinocyte proliferation. The EF-hand calcium binding protein S100A7 has been found to be overexpressed in psoriatic keratinocytes. It is know that S100A7 may interact with Jab1, a cofactor that stabilizes c-Jun. Jab1 is known to downregulate the expression of the cell cycle inhibitor p27^Kip1 in some cancer models. In this study, we aimed to investigate the possible interaction between S100A7 and Jab1 and the downstream effects on p27 ^Kip1 expression in normal human keratinocyte cells transfected with S100A7 CRISPR activation plasmid and in archival psoriatic skin samples. Our results showed that the upregulated S100A7 colocalizes with Jab1 at the nuclear level in transfected cells and psoriatic skin samples. We also showed a differential protein expression of Jab1 between cytoplasmic and nuclear compartments, thus suggesting Jab1 translocation from nucleus to cytoplasm. p27 ^Kip1 protein expression patterns would imply a translocation from nucleus and a subsequent degradation of this protein. The upregulation of S1007 and its interaction with Jab1 would contribute to the p27 ^Kip1-dependent impaired proliferation that characterizes psoriatic skin.     

MiR-1-3p通过抑制CAPRIN1调控胰腺癌发生发展

摘要 目的：探讨miR-1-3p在胰腺癌发生发展中的分子机制。方法：以MIA-PaCa-2,SW 1990为研究目标,通过qRT-PCR技术检测miR-1-3p的表达量,利用TargetScan和miRDB数据库预测miR-1-3p的下游靶基因及结合位点,并通过构建双荧光素酶报告基因,进一步确认miR-1-3p与靶基因的结合。利用CCK8细胞增殖实验及平板克隆形成实验检测过表达miR-1-3p及敲低CAPRIN1对细胞增殖的作用;利用流式检测细胞周期;利用蛋白质免疫印迹方法检测miR-1-3p对CAPRIN1及其下游基因的影响;通过流式来确认,过表达miR-1-3p及敲减CAPRIN1基因对细胞周期的影响。结果：miR-1-3p在胰腺癌细胞MIA-PaCa-2,SW 1990中低表达;miR-1-3p直接与CAPRIN1的3''-untranslated region (3''- UTR)结合;过表达miR-1-3p或抑制CAPRIN1基因的表达可明显抑制胰腺癌细胞的增殖能力,同时也产生细胞周期阻滞。结论：miR-1-3p通过抑制CAPRIN1基因表达,而产生细胞周期阻滞进而抑制胰腺癌细胞的增殖能力。     

Comprehensive analysis of cellular galectin-3 reveals no consistent oncogenic function in pancreatic cancer cells

Galectin-3 (Gal-3), a 31 kDa member of the family of beta-galactoside-binding proteins, has been implicated in the progression of different human cancers. However, the proposed roles differ widely, ranging from tumor-promoting cellular functions and negative impact on patient prognosis to tumor-suppressive properties and positive prognostic impact. We and others have previously identified Gal-3 as overexpressed in pancreatic cancer as compared to chronic pancreatitis and normal pancreatic tissue. The purpose of this study was thus the comprehensive analysis of putative cellular functions of Gal-3 by transient as well as stable silencing or overexpression of Gal-3 in a panel of 6 well-established pancreatic cancer cell lines. Our results confirm that galectin-3 is upregulated at the mRNA level in pancreatic cancer and strongly expressed in the majority of pancreatic cancer cell lines. In individual cell lines, transient knockdown of Gal-3 expression resulted in moderate inhibitory effects on proliferation, migration or anchorage-independent growth of the cells, but these effects were not consistent across the spectrum of analyzed cell lines. Moreover, functional effects of the modulation of Gal-3 expression were not observed in stable knockdown or overexpression approaches in vitro and did not alter the growth characteristics of nude mouse xenograft tumors in vivo. Our data thus do not support a direct functional role of Gal-3 in the malignant transformation of pancreatic epithelial cells, although paracrine or systemic effects of Gal-3 expression are not excluded.     

Molecular consequences of silencing mutant K-ras in pancreatic cancer cells: justification for K-ras-directed therapy

Mutation of the K-ras gene is an early event in the development of pancreatic adenocarcinoma and, therefore, RNA interference (RNAi) directed toward mutant K-ras could represent a novel therapy. In this study, we examine the phenotypic and molecular consequences of exposure of pancreatic tumor cells to mutant-specific K-ras small interfering RNA. Specific reduction of activated K-ras via RNAi in Panc-1 and MiaPaca-2 cells resulted in cellular changes consistent with a reduced capacity to form malignant tumors. These changes occur through distinct mechanisms but likely reflect an addiction of each cell line to oncogene stimulation. Both cell lines show reduced proliferation after K-ras RNAi, but only MiaPaca-2 cells showed increased apoptosis. Both cell lines showed reduced migration after K-ras knockdown, but changes in integrin levels were not consistent between the cell lines. Both cell lines showed alteration of the level of GLUT-1, a metabolism-associated gene that is downstream of c-myc, with Panc-1 cells demonstrating decreased GLUT-1 levels, whereas MiaPaca-2 cells showed increased levels of expression after K-ras knockdown. Furthermore, after K-ras RNAi, there was a reduction in angiogenic potential of both Panc-1 and MiaPaca-2 cells. Panc-1 cells increased the level of expression of thrombospondin-1, an endogenous inhibitor of angiogenesis, whereas MiaPaca-2 cells decreased the production of vascular endothelial growth factor, a primary stimulant of angiogenesis in pancreatic tumors. We have found that silencing mutant K-ras through RNAi results in alteration of tumor cell behavior in vitro and suggests that targeting mutant K-ras specifically might be effective against pancreatic cancer in vivo.     

Lipoxygenase inhibitors abolish proliferation of human pancreatic cancer cells.

Epidemiologic and animal studies have linked pancreatic cancer growth with fat intake, especially unsaturated fats. Arachidonic acid release from membrane phospholipids is essential for tumor cell proliferation. Lipoxygenases (LOX) constitute one pathway for arachidonate metabolism, but their role in pancreatic cancer growth is unknown. The expression of 5-LOX and 12-LOX as well as their effects on cell proliferation was investigated in four human pancreatic cancer cell lines (PANC-1, MiaPaca2, Capan2, and ASPC-1). Expression of 5-LOX and 12-LOX mRNA was measured by nested RT-PCR. Effects of LOX inhibitors and specific LOX antisense oligonucleotides on pancreatic cancer cell proliferation were measured by (3)H-thymidine incorporation. Our results showed that (1) 5-LOX and 12-LOX were expressed in all pancreatic cancer cell lines tested, while they were not detectable in normal human pancreatic ductal cells; (2) both LOX inhibitors and LOX antisense markedly inhibited cell proliferation in a concentration-dependent and time-dependent manner; (3) the 5-LOX and 12-LOX metabolites 5-HETE and 12-HETE as well as arachidonic and linoleic acids directly stimulated pancreatic cancer cell proliferation; (4) LOX inhibitor-induced growth inhibition was reversed by 5-HETE and 12-HETE. The current studies indicate that both 5-LOX and 12-LOX expression is upregulated in human pancreatic cancer cells and LOX plays a critical role in pancreatic cancer cell proliferation. LOX inhibitors may be valuable for the treatment of pancreatic cancer.     

Deregulated c-myc expression abrogates the interferon- and interleukin 6-mediated G0/G1 cell cycle arrest but not other inhibitory responses in M1 myeloblastic cells.

The proliferation of M1 myeloblastic cells can be specifically restricted at the G0/G1 phase of the cell cycle by exposure to alpha- and beta-interferons or to interleukin 6. The latter cytokine also induces the morphological and functional differentiation of these myeloblasts toward monocytes. Each of these two different cytokines suppresses the expression of the c-myc nuclear oncogene, and the selective reduction in c-myc mRNA and protein precedes the cell cycle changes. In order to investigate whether one or more of the growth-suppressive effects of interferon and interleukin 6 are mediated by c-myc reduction, M1 cells were transfected with SV40-driven c-myc plasmid, whose expression fails to be turned off by these two cytokines. A detailed analysis of the responses to interferon and to interleukin 6 revealed that all of the myc-transfected clones have lost the cytokine-mediated G0/G1 type of growth arrest. However, not all of the growth responses to these cytokines were rescued by this specific genetic manipulation, and the cytokine-treated transfected cells stopped to proliferate in a new fashion which was not cell cycle specific. In addition, the myc-transfected cells developed the differentiated phenotype in response to interleukin 6, as determined by the morphological change, expression of Fc receptors, and cytochemical analysis, suggesting that these molecular events can occur in the monocyte cell lineage in spite of the abnormal constitutive expression of c-myc.(ABSTRACT TRUNCATED AT 250 WORDS)     

Gfer inhibits Jab1-mediated degradation of p27kip1 to restrict proliferation of hematopoietic stem cells

Growth factor erv1-like (Gfer) is an evolutionarily conserved sulfhydryl oxidase that is enriched in embryonic and adult stem cells and plays an essential prosurvival role in pluripotent embryonic stem cells. Here we show that knockdown (KD) of Gfer in hematopoietic stem cells (HSCs) compromises their in vivo engraftment potential and triggers a hyper-proliferative response that leads to their exhaustion. KD of Gfer in HSCs does not elicit a significant alteration of mitochondrial morphology or loss of cell viability. However, these cells possess significantly reduced levels of the cyclin-dependent kinase inhibitor p27(kip1). In contrast, overexpression of Gfer in HSCs results in significantly elevated total and nuclear p27(kip1). KD of Gfer results in enhanced binding of p27(kip1) to its inhibitor, the COP9 signalosome subunit jun activation-domain binding protein 1 (Jab1), leading to its down-regulation. Conversely, overexpression of Gfer results in its enhanced binding to Jab1 and inhibition of the Jab1-p27(kip1) interaction. Furthermore, normalization of p27(kip1) in Gfer-KD HSCs rescues their in vitro proliferation deficits. Taken together, our data demonstrate the presence of a novel Gfer-Jab1-p27(kip1) pathway in HSCs that functions to restrict abnormal proliferation.     

GATA6 activates Wnt signaling in pancreatic cancer by negatively regulating the Wnt antagonist Dickkopf-1

Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal disease characterized by late diagnosis and treatment resistance. Recurrent genetic alterations in defined genes in association with perturbations of developmental cell signaling pathways have been associated with PDAC development and progression. Here, we show that GATA6 contributes to pancreatic carcinogenesis during the temporal progression of pancreatic intraepithelial neoplasia by virtue of Wnt pathway activation. GATA6 is recurrently amplified by both quantitative-PCR and fluorescent in-situ hybridization in human pancreatic intraepithelial neoplasia and in PDAC tissues, and GATA6 copy number is significantly correlated with overall patient survival. Forced overexpression of GATA6 in cancer cell lines enhanced cell proliferation and colony formation in soft agar in vitro and growth in vivo, as well as increased Wnt signaling. By contrast siRNA mediated knockdown of GATA6 led to corresponding decreases in these same parameters. The effects of GATA6 were found to be due to its ability to bind DNA, as forced overexpression of a DNA-binding mutant of GATA6 had no effects on cell growth in vitro or in vivo, nor did they affect Wnt signaling levels in these same cells. A microarray analysis revealed the Wnt antagonist Dickopf-1 (DKK1) as a dysregulated gene in association with GATA6 knockdown, and direct binding of GATA6 to the DKK1 promoter was confirmed by chromatin immunoprecipitation and electrophoretic mobility shift assays. Transient transfection of GATA6, but not mutant GATA6, into cancer cell lines led to decreased DKK1 mRNA expression and secretion of DKK1 protein into culture media. Forced overexpression of DKK1 antagonized the effects of GATA6 on Wnt signaling in pancreatic cancer cells. These findings illustrate that one mechanism by which GATA6 promotes pancreatic carcinogenesis is by virtue of its activation of canonical Wnt signaling via regulation of DKK1.