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1.  Multifunctional roles of urokinase plasminogen activator (uPA) in cancer stemness and chemoresistance of pancreatic cancer 
Molecular Biology of the Cell  2013;24(17):2620-2632.
This research highlights the multifunctional role of uPA in pancreatic cancer as a regulator of stemness and chemoresistance. uPA is found to be physically associated with the transcription factors Lhx2 and HOXA5 in the nucleus and regulated expression of p53.
Pancreatic ductal adenocarcinoma (PDAC) is almost always lethal. One of the underlying reasons for this lethality is believed to be the presence of cancer stem cells (CSC), which impart chemoresistance and promote recurrence, but the mechanisms responsible are unclear. Recently the poor prognosis of PDAC has been correlated with increased expression of urokinase plasminogen activator (uPA). In the present study we examine the role of uPA in the generation of PDAC CSC. We observe a subset of cells identifiable as a side population (SP) when sorted by flow cytometry of MIA PaCa-2 and PANC-1 pancreatic cancer cells that possess the properties of CSC. A large fraction of these SP cells are CD44 and CD24 positive, are gemcitabine resistant, possess sphere-forming ability, and exhibit increased tumorigenicity, known characteristics of cancer stemness. Increased tumorigenicity and gemcitabine resistance decrease after suppression of uPA. We observe that uPA interacts directly with transcription factors LIM homeobox-2 (Lhx2), homeobox transcription factor A5 (HOXA5), and Hey to possibly promote cancer stemness. uPA regulates Lhx2 expression by suppressing expression of miR-124 and p53 expression by repressing its promoter by inactivating HOXA5. These results demonstrate that regulation of gene transcription by uPA contributes to cancer stemness and clinical lethality.
PMCID: PMC3756915  PMID: 23864708
2.  Updated Analysis of SWOG-Directed Intergroup Study 0116: A Phase III Trial of Adjuvant Radiochemotherapy Versus Observation After Curative Gastric Cancer Resection 
Journal of Clinical Oncology  2012;30(19):2327-2333.
Surgical resection of gastric cancer has produced suboptimal survival despite multiple randomized trials that used postoperative chemotherapy or more aggressive surgical procedures. We performed a randomized phase III trial of postoperative radiochemotherapy in those at moderate risk of locoregional failure (LRF) following surgery. We originally reported results with 4-year median follow-up. This update, with a more than 10-year median follow-up, presents data on failure patterns and second malignancies and explores selected subset analyses.
Patients and Methods
In all, 559 patients with primaries ≥ T3 and/or node-positive gastric cancer were randomly assigned to observation versus radiochemotherapy after R0 resection. Fluorouracil and leucovorin were administered before, during, and after radiotherapy. Radiotherapy was given to all LRF sites to a dose of 45 Gy.
Overall survival (OS) and relapse-free survival (RFS) data demonstrate continued strong benefit from postoperative radiochemotherapy. The hazard ratio (HR) for OS is 1.32 (95% CI, 1.10 to 1.60; P = .0046). The HR for RFS is 1.51 (95% CI, 1.25 to 1.83; P < .001). Adjuvant radiochemotherapy produced substantial reduction in both overall relapse and locoregional relapse. Second malignancies were observed in 21 patients with radiotherapy versus eight with observation (P = .21). Subset analyses show robust treatment benefit in most subsets, with the exception of patients with diffuse histology who exhibited minimal nonsignificant treatment effect.
Intergroup 0116 (INT-0116) demonstrates strong persistent benefit from adjuvant radiochemotherapy. Toxicities, including second malignancies, appear acceptable, given the magnitude of RFS and OS improvement. LRF reduction may account for the majority of overall relapse reduction. Adjuvant radiochemotherapy remains a rational standard therapy for curatively resected gastric cancer with primaries T3 or greater and/or positive nodes.
PMCID: PMC4517071  PMID: 22585691
3.  Diagnostic accuracy of endoscopic ultrasound in pancreatic neuroendocrine tumors: A systematic review and meta analysis 
AIM: To detect pancreatic neuroendocrine tumors (PNETs) has been varied. This study is undertaken to evaluate the accuracy of endoscopic ultrasound (EUS) in detecting PNETs.
METHODS: Only EUS studies confirmed by surgery or appropriate follow-up were selected. Articles were searched in Medline, Ovid journals, Medline nonindexed citations, and Cochrane Central Register of Controlled Trials and Database of Systematic Reviews. Pooling was conducted by both fixed and random effects model).
RESULTS: Initial search identified 2610 reference articles, of these 140 relevant articles were selected and reviewed. Data was extracted from 13 studies (n = 456) which met the inclusion criteria. Pooled sensitivity of EUS in detecting a PNETs was 87.2% (95%CI: 82.2-91.2). EUS had a pooled specificity of 98.0% (95%CI: 94.3-99.6). The positive likelihood ratio of EUS was 11.1 (95%CI: 5.34-22.8) and negative likelihood ratio was 0.17 (95%CI: 0.13-0.24). The diagnostic odds ratio, the odds of having anatomic PNETs in positive as compared to negative EUS studies was 94.7 (95%CI: 37.9-236.1). Begg-Mazumdar bias indicator for publication bias gave a Kendall’s tau value of 0.31 (P = 0.16), indication no publication bias. The P for χ2 heterogeneity for all the pooled accuracy estimates was > 0.10.
CONCLUSION: EUS has excellent sensitivity and specificity to detect PNETs. EUS should be strongly considered for evaluation of PNETs.
PMCID: PMC3691045  PMID: 23801872
Endoscopic ultrasound; Ultrasound; Endosonography; Pancreatic mass; Neuroendocrine tumors; Sensitivity; Specificity; Positive predictive value; Negative predictive value
4.  Localization of uPAR and MMP-9 in lipid rafts is critical for migration, invasion and angiogenesis in human breast cancer cells 
BMC Cancer  2010;10:647.
uPAR and MMP-9, which play critical roles in tumor cell invasion, migration and angiogenesis, have been shown to be associated with lipid rafts.
To investigate whether cholesterol could regulate uPAR and MMP-9 in breast carcinoma, we used MβCD (methyl beta cyclodextrin, which extracts cholesterol from lipid rafts) to disrupt lipid rafts and studied its effect on breast cancer cell migration, invasion, angiogenesis and signaling.
Morphological evidence showed the association of uPAR with lipid rafts in breast carcinoma cells. MβCD treatment significantly reduced the colocalization of uPAR and MMP-9 with lipid raft markers and also significantly reduced uPAR and MMP-9 at both the protein and mRNA levels. Spheroid migration and invasion assays showed inhibition of breast carcinoma cell migration and invasion after MβCD treatment. In vitro angiogenesis studies showed a significant decrease in the angiogenic potential of cells pretreated with MβCD. MβCD treatment significantly reduced the levels of MMP-9 and uPAR in raft fractions of MDA-MB-231 and ZR 751 cells. Phosphorylated forms of Src, FAK, Cav, Akt and ERK were significantly inhibited upon MβCD treatment. Increased levels of soluble uPAR were observed upon MβCD treatment. Cholesterol supplementation restored uPAR expression to basal levels in breast carcinoma cell lines. Increased colocalization of uPAR with the lysosomal marker LAMP1 was observed in MβCD-treated cells when compared with untreated cells.
Taken together, our results suggest that cholesterol levels in lipid rafts are critical for the migration, invasion, and angiogenesis of breast carcinoma cells and could be a critical regulatory factor in these cancer cell processes mediated by uPAR and MMP-9.
PMCID: PMC3002355  PMID: 21106094
5.  Small Interfering RNA (siRNA)-Directed Knockdown of Uracil DNA Glycosylase Induces Apoptosis and Sensitizes Human Prostate Cancer Cells to Genotoxic Stress 
Molecular cancer research : MCR  2009;7(8):1285-1293.
Uracil DNA glycosylase (UNG) is the primary enzyme responsible for removing uracil residues from DNA. Although a substantial body of evidence suggests that DNA damage plays a role in cancer cell apoptosis, the underlying mechanisms are poorly understood. In particular, very little is known about the role of base excision repair of misincorporated uracil in cell survival. To test the hypothesis that the repair of DNA damage associated with uracil misincorporation is critical for cancer cell survival, we used small interfering RNA to target the human UNG gene. In a dose- and time-dependent manner, siRNA specifically inhibited UNG expression and modified expression of several genes, at both mRNA and protein levels. In LNCaP cells, p53, p21 and Bax protein levels increased, whereas Bcl2 levels decreased. In DU145 cells, p21 levels were elevated, although mutant p53 and Bax levels remained unchanged. In PC3 cells, UNG inhibition resulted in elevated p21 and Bax levels. In all three cell lines, UNG inhibition reduced cell proliferation, induced apoptosis, and increased cellular sensitivity to genotoxic stress. Furthermore, the in vitro cleavage experiment using uracil-containing double-stranded DNA as template has demonstrated that siRNA-mediated knockdown of UNG expression significantly reduced uracil-excising activity of UNG in human prostate cancer cells, which was associated with DNA damage analyzed by comet assay. Taken together, these findings indicate that RNA interference-directed targeting of UNG is a convenient, novel tool for studying the biological role of UNG and raises the potential of its application for prostate cancer therapy.
PMCID: PMC2730435  PMID: 19671688
Uracil DNA glycosylase; DNA repair; apoptosis; prostate cancer
6.  Stat3-siRNA Induces Fas-mediated Apoptosis in vitro and in vivo in Breast Cancer 
International journal of oncology  2009;34(5):1209-1220.
Stat3, a member of the signal transducer and activator of transcription family, has the potential to mediate cell survival, growth and differentiation. Stat3 is constitutively activated in numerous cancers, including more than 50% of breast cancers. Previous studies demonstrated that constitutively activated Stat3 plays an important role in breast cancer development and progression by promoting cell proliferation and inhibiting apoptosis. The present study was designed to investigate the potential use of RNA interference (RNAi) to block Stat3 expression and activation, as well as the subsequent effect on human breast cancer cell growth. Our studies show that knockdown of STAT3 expression by siRNA reduced expression of Bcl-xL and survivin in MDA-MB-231 cells, and also led to Fas mediated intrinsic apoptotic pathway by activating Caspases -8, -9, -3 and PARP 1 cleavage. In nude mice, pRNAi-Stat3 significantly suppressed tumor growth compared with controls. It also suppressed Stat3 expression, and downregulated BcL-xL and upregulated Fas, Fas-L and cleaved Caspase 3 expression within the tumor, which significantly induced apoptosis and led to tumor suppression. Thus, targeting Stat3 signaling using siRNA may serve as a novel therapeutic strategy for the treatment of breast cancers expressing constitutively activated Stat3.
PMCID: PMC2668130  PMID: 19360334
Stat3; apoptosis; Fas/Fas-L
7.  MMP-9 Inhibition Downregulates Radiation-induced NF-κ Activity Leading to Apoptosis in Breast Tumors 
Novel strategies are needed to prevent the high mortality rates of several types of cancer. These high rates stem from tumor resistance to radiation therapy, which is thought to result from the induction of matrix metalloproteinases and plasminogen activators. In the present study, we show that the modulation of MMP-9 expression, using adenoviral-mediated transfer of the antisense MMP-9 gene (Ad-MMP-9), affects breast cancer sensitivity to radiation.
Experimental Design
In the present study, we used antisense MMP-9 adenoviral construct (Ad-MMP-9) to downregulate the expression of MMP-9 in MDA MB 231 breast cancer cell lines in vitro prior to irradiation and subsequently incubated cells in hypoxic condition. In vivo studies were performed with orthotopic breast tumors and the radiosensitivity evaluated both in vitro and in vivo.
Ad-MMP-9 infection resulted in downregulation of radiation-induced levels of hypoxia-inducible factor 1 alpha (HIF1α) and MMP-9 under hypoxic conditions in MDA MB 231 breast cancer cells. In addition, Ad-MMP-9 in combination with radiation decreased levels of the transcription factors NF-κB and AP 1, both of which contribute to the radioresistance of breast tumors. Finally, the triggering of the Fas-Fas-L apoptotic cascade, which resulted in the cleavage of PARP-1 and caspases 10, 3 and 7, signifies the efficiency of combined treatment of Ad-MMP-9 and radiation. Treatment with Ad-MMP-9 plus radiation completely regressed tumor growth in orthotopic breast cancer model.
In summary, integrating gene therapy (adenovirus-mediated inhibition of MMP-9) with radiotherapy could have a synergistic effect, thereby improving the survival of patients with breast cancer.
PMCID: PMC2410036  PMID: 18519796
Ad-MMP-9; Radiation; Orthotopic breast tumor; Hypoxia; Tumor growth
8.  RNAi-mediated Downregulation of Urokinase Plasminogen Activator Receptor (uPAR) and Matrix Metalloprotease-9 (MMP-9) in Human Breast Cancer Cells Results in Decreased Tumor Invasion, Angiogenesis and Growth 
The serine protease urokinase-type plasminogen activator (uPA) plays a significant role in tumor cell invasion and metastasis when bound to its specific receptor, uPAR (also known as CD87). In addition to the uPA-uPAR system, matrix metalloproteinases (MMPs) are involved in tumor cell invasion and metastasis. In this study, we achieved specific inhibition of uPAR and MMP-9 using RNAi technology. We introduced small interfering RNA (siRNA) to downregulate the expression of uPAR and MMP-9 (pUM) in breast cancer cell lines (MDA MB 231 and ZR 75 1). In vitro angiogenesis studies indicated a decrease in the angiogenic potential of the treated cells; in particular, a remarkable decrease was observed in the cells treated with bicistronic construct (pUM) in comparision to the controls. Additionally, bicistronic construct inhibited the formation of capillary-like structures in in vivo models of angiogenesis. Similarly, the invasive potential and migration decreased dramatically when treated with the bicistronic construct as shown by matrigel invasion and migration assays. These results suggest a synergistic effect from the simultaneous downregulation of uPAR and MMP-9. We also assessed the levels of phosphorylated forms of MAPK, ERK, and AKT signaling pathway molecules and found reduction in the levels of these molecules in cells treated with the bicistronic construct as compared to the control cells. Furthermore, targeting both uPAR and MMP-9 totally regressed orthotopic breast tumors in nude mice. In conclusion, our results provide evidence that the simultaneous downregulation of uPAR and MMP-9 using RNAi technology may provide an effective tool for breast cancer therapy.
PMCID: PMC2396459  PMID: 17657740
RNAi; uPAR; MMP-9; Invasion; Angiogenesis; Tumor growth
9.  Epigenetic Inactivation of the Tissue Inhibitor of Metalloproteinase-2 (TIMP-2) Gene in Human Prostate Tumors 
Oncogene  2007;26(36):5229-5237.
Gene silencing via CpG island methylation in the promoter region is one of the mechanisms by which tumor suppressor genes are inactivated in human cancers. Previous studies have shown that the TIMP-2 gene, which is an endogenous inhibitor of matrix metalloproteinases involved in cell invasion and tumorigenesis, is downregulated or silenced in a variety of human cancer cell lines. Here, we investigated the mechanism underlying TIMP-2 expression in prostate cancer cell lines and primary prostate tumor samples. We observed a strong correlation between promoter hypermethylation and lost expression of TIMP-2 gene, which was supported by other results demonstrating that promoter demethylation by 5-aza-2′-deoxycytidine and trichostatin A reactivated TIMP-2 and restored its expression in TIMP-2-silenced metastatic prostate cell lines. These result were further substantiated by a chromatin immunoprecipitation assay, showing the preferential binding of MeCP2 to methylated CpG island in TIMP-2-silenced metastatic prostate cell lines. In vitro Matrigel invasion assays showed that reexpression of TIMP-2 after a combined treatment with 5-aza and TSA in metastatic prostate cells resulted in a significant reduction of tumor cell invasion. Furthermore, CpG methylation of TIMP-2 promoter was also shown in primary prostate tumors that expressed decreased TIMP-2 protein levels. These results suggest that the downregulation of the TIMP-2 gene is associated with promoter methylation and that this may play an important role in prostate cancer progression during the invasive and metastatic stages of the disease.
PMCID: PMC2084378  PMID: 17325663
CpG methylation; TIMP-2; MeCP2; Prostate cancer
10.  Demethylation-linked Activation of uPA is Involved in Progression of Prostate Cancer 
Cancer research  2007;67(3):930-939.
Increased expression of urokinase plasminogen activator (uPA) has been reported in various malignancies including prostate cancer. However, the mechanism by which uPA is abnormally expressed in prostate cancer remains elusive. Here, we show that uPA is aberrantly expressed in a high-percentage of human prostate cancer tissues, but rarely expressed either in tumor-matched, non-neoplastic adjacent tissues (NNAT) or benign prostatic hyperplasia (BPH) samples. This aberrant expression is associated with cancer-linked demethylation of the uPA promoter. Furthermore, treatment with demethylation inhibitor S-Adenosylmethionine (Ado-Met) or stable expression of uPA shRNA significantly inhibits uPA expression and tumor cell invasion in vitro and tumor growth and incidence of lung metastasis in vivo. Collectively, these findings strongly suggest that DNA demethylation is a common mechanism underlying the abnormal expression of uPA and is a critical contributing factor to the malignant progression of human prostate tumors.
PMCID: PMC1832148  PMID: 17283123
DNA Demethylation; Metastasis; Prostate cancer; Urokinase Plasminogen Activator
11.  RNA Interference-Directed Knockdown of Urokinase Plasminogen Activator and Urokinase Plasminogen Activator Receptor Inhibits Prostate Cancer Cell Invasion, Survival and Tumorigenicity In vivo 
The Journal of biological chemistry  2005;280(43):36529-36540.
The invasive ability of tumor cells plays a key role in prostate cancer metastasis and is a major cause of treatment failure. Urokinase plasminogen activator (uPA) and its receptor (uPAR)-mediated signaling have been implicated in tumor cell invasion, survival and metastasis in a variety of cancers. This study was undertaken to investigate the biological roles of uPA and uPAR in prostate cancer cell invasion, survival and the potential of uPA and uPAR as targets for prostate cancer therapy. uPA and uPAR expression correlates with the metastatic potential of prostate cancer cells. Thus, therapies designed to inhibit uPA and uPAR expression would be beneficial. LNCaP, DU145 and PC3 are prostate cancer cell lines with low, moderate and high metastatic potential, respectively, as demonstrated by their capacity to invade extracellular matrix (ECM). In this study we utilized small hairpin RNAs (shRNAs), also referred to as small interfering RNAs (siRNAs), to target human uPA and uPAR. These siRNA constructs significantly inhibited uPA and uPAR expression at both the mRNA and protein levels in the highly metastatic prostate cancer cell line PC3. Our data demonstrate that uPA-uPAR knockdown in PC3 cells resulted in a dramatic reduction of tumor cell invasion as indicated by a matrigel invasion assay. Furthermore, simultaneous silencing of the genes for uPA and uPAR using a single plasmid construct expressing shRNAs for both uPA and uPAR significantly reduced cell viability and ultimately resulted in the induction of apoptotic cell death. RNAi for uPA and uPAR also abrogated uPA-uPAR signaling to downstream target molecules such as extracellular-signal regulated kinases 1/2 (ERK1/2) and the signal transducer and activator of transcription 3 (Stat 3). In addition, our results demonstrate that intratumoral injection with the plasmid construct expressing shRNAs for uPA and uPAR almost completely inhibited established tumor growth and survival in an orthotopic mouse prostate cancer model. These findings uncovered evidence of a complex signaling network operating downstream of uPA-uPAR that actively advances tumor cell invasion, proliferation and survival of prostate cancer cells. Thus, RNAi-directed targeting of uPA and uPAR is a convenient and novel tool for studying the biological role of the uPA-uPAR system and raises the potential of its application for prostate cancer therapy.
PMCID: PMC1351057  PMID: 16127174
RNAi; prostate cancer; invasion; survival; uPA and uPAR; siRNA (small interfering RNA); shRNA (small hairpin RNA); RNAi (RNA interference); uPA (urokinaseplasminogen activator); uPAR (uPA receptor); ANOVA (analysis of variance); RT (reverse transcription); TPBS (tween-20 (0.1%) phospate buffered saline); BSA (bovine serum albumin)

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