Dejuan Kong

miR-200 Regulates PDGF-D-Mediated Epithelial-Mesenchymal Transition, Adhesion, and Invasion of Prostate Cancer Cells

MicroRNAs have been implicated in tumor progression. Recent studies have shown that the miR‐200 family regulates epithelial–mesenchymal transition (EMT) by targeting zinc‐finger E‐box binding homeobox 1 (ZEB1) and ZEB2. Emerging evidence from our laboratory and others suggests that the processes of EMT can be triggered by various growth factors, such as transforming growth factor β and platelet‐derived growth factor‐D (PDGF‐D). Moreover, we recently reported that overexpression of PDGF‐D in prostate cancer cells (PC3 PDGF‐D cells) leads to the acquisition of the EMT phenotype, and this model offers an opportunity for investigating the molecular interplay between PDGF‐D signaling and EMT. Here, we report, for the first time, significant downregulation of the miR‐200 family in PC3 PDGF‐D cells as well as in PC3 cells exposed to purified active PDGF‐D protein, resulting in the upregulation of ZEB1, ZEB2, and Snail2 expression. Interestingly, re‐expression of miR‐200b in PC3 PDGF‐D cells led to reversal of the EMT phenotype, which was associated with the downregulation of ZEB1, ZEB2, and Snail2 expression, and these results were consistent with greater expression levels of epithelial markers. Moreover, transfection of PC3 PDGF‐D cells with miR‐200b inhibited cell migration and invasion, with concomitant repression of cell adhesion to the culture surface and cell detachment. From these results, we conclude that PDGF‐D‐induced acquisition of the EMT phenotype in PC3 cells is, in part, a result of repression of miR‐200 and that any novel strategy by which miR‐200 could be upregulated would become a promising approach for the treatment of invasive prostate cancer. STEM CELLS 2009;27:1712–1721

Targeting miRNAs involved in cancer stem cell and EMT regulation: An emerging concept in overcoming drug resistance

Although chemotherapy is an important therapeutic strategy for cancer treatment, it fails to eliminate all tumor cells due to intrinsic or acquired drug resistance, which is the most common cause of tumor recurrence. Emerging evidence suggests an intricate role of cancer stem cells (CSCs) and epithelial-mesenchymal transition (EMT)-type cells in anticancer drug resistance. Recent studies also demonstrated that microRNAs (miRNAs) play critical roles in the regulation of drug resistance. Here we will discuss current knowledge regarding CSCs, EMT and the role of regulation by miRNAs in the context of drug resistance, tumor recurrence and metastasis. A better understanding of the molecular intricacies of drug-resistant cells will help to design novel therapeutic strategies by selective targeting of CSCs and EMT-phenotypic cells through alterations in the expression of specific miRNAs towards eradicating tumor recurrence and metastasis. A particular promising lead is the potential synergistic combination of natural compounds that affect critical miRNAs, such as curcumin or epigallocatechin-3-gallate (EGCG) with chemotherapeutic agents.

Notch-1 induces Epithelial-mesenchymal transition consistent with cancer stem cell phenotype in pancreatic cancer cells

Activation of Notch-1 is known to be associated with the development and progression of human malignancies including pancreatic cancer. Emerging evidence suggest that the acquisition of epithelial-mesenchymal transition (EMT) phenotype and induction of cancer stem cell (CSC) or cancer stem-like cell phenotype are interrelated and contributes to tumor recurrence and drug resistance. The molecular mechanism(s) by which Notch-1 contributes to the acquisition of EMT phenotype and CSC self-renewal capacity has not been fully elucidated. Here we show that forced over-expression of Notch-1 leads to increased cell growth, clonogenicity, migration and invasion of AsPC-1 cells. Moreover, over-expression of Notch-1 led to the induction of EMT phenotype by activation of mesenchymal cell markers such as ZEB1, CD44, EpCAM, and Hes-1. Here we also report, for the first time, that over-expression of Notch-1 leads to increased expression of miR-21, and decreased expression of miR-200b, miR-200c, let-7a, let-7b, and let-7c. Re-expression of miR-200b led to decreased expression of ZEB1, and vimentin, and increased expression of E-cadherin. Over-expression of Notch-1 also increased the formation of pancreatospheres consistent with expression of CSC surface markers CD44 and EpCAM. Finally, we found that genistein, a known natural anti-tumor agent inhibited cell growth, clonogenicity, migration, invasion, EMT phenotype, formation of pancreatospheres and expression of CD44 and EpCAM. These results suggest that the activation of Notch-1 signaling contributes to the acquisition of EMT phenotype, which is in part mediated through the regulation of miR-200b and CSC self-renewal capacity, and these processes could be attenuated by genistein treatment.

Cancer Stem Cells and Epithelial-to-Mesenchymal Transition (EMT)-Phenotypic Cells: Are They Cousins or Twins?

Cancer stem cells (CSCs) are cells within a tumor that possess the capacity to self-renew and maintain tumor-initiating capacity through differentiation into the heterogeneous lineages of cancer cells that comprise the whole tumor. These tumor-initiating cells could provide a resource for cells that cause tumor recurrence after therapy. Although the cell origin of CSCs remains to be fully elucidated, mounting evidence has demonstrated that Epithelial-to-Mesenchymal Transition (EMT), induced by different factors, is associated with tumor aggressiveness and metastasis and these cells share molecular characteristics with CSCs, and thus are often called cancer stem-like cells or tumor-initiating cells. The acquisition of an EMT phenotype is a critical process for switching early stage carcinomas into invasive malignancies, which is often associated with the loss of epithelial differentiation and gain of mesenchymal phenotype. Recent studies have demonstrated that EMT plays a critical role not only in tumor metastasis but also in tumor recurrence and that it is tightly linked with the biology of cancer stem-like cells or cancer-initiating cells. Here we will succinctly summarize the state-of-our-knowledge regarding the molecular similarities between cancer stem-like cells or CSCs and EMT-phenotypic cells that are associated with tumor aggressiveness focusing on solid tumors.

Down-regulation of Forkhead Box M1 Transcription Factor Leads to the Inhibition of Invasion and Angiogenesis of Pancreatic Cancer Cells

The Forkhead Box M1 (FoxM1) transcription factor has been shown to play important roles in regulating the expression of genes involved in cell proliferation, differentiation, and transformation. Overexpression of FoxM1 has been found in a variety of aggressive human carcinomas including pancreatic cancer. However, the precise role and the molecular mechanism of action of FoxM1 in pancreatic cancer remain unclear. To elucidate the cellular and molecular function of FoxM1, we tested the consequences of down-regulation and up-regulation of FoxM1 in pancreatic cancer cells, respectively. Using multiple cellular and molecular approaches such as 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, gene transfection, flow cytometry, real-time reverse transcription-PCR, Western blotting, migration, invasion, and angiogenesis assays, we found that down-regulation of FoxM1 inhibited cell growth, decreased cell migration, and decreased invasion of pancreatic cancer cells. FoxM1 down-regulation also decreased cell population in the S phase. Compared with control, FoxM1 small interfering RNA-transfected cells showed decreased expression of cyclin B, cyclin D1, and Cdk2, whereas p21 and p27 expression was increased. We also found that down-regulation of FoxM1 reduced the expression of matrix metalloproteinase-2 (MMP-2), MMP-9 and vascular endothelial growth factor, resulting in the inhibition of migration, invasion, and angiogenesis. These findings suggest that FoxM1 down-regulation could be a novel approach for the inhibition of pancreatic tumor progression.

Metformin Inhibits Cell Proliferation, Migration and Invasion by Attenuating CSC Function Mediated by Deregulating miRNAs in Pancreatic Cancer Cells

Pancreatic cancer is the fourth leading cause of cancer-related deaths in the United States, which is, in part, due to intrinsic (de novo) and extrinsic (acquired) resistance to conventional therapeutics, suggesting that innovative treatment strategies are required for overcoming therapeutic resistance to improve overall survival of patients. Oral administration of metformin in patients with diabetes mellitus has been reported to be associated with reduced risk of pancreatic cancer and that metformin has been reported to kill cancer stem cells (CSC); however, the exact molecular mechanism(s) has not been fully elucidated. In the current study, we examined the effect of metformin on cell proliferation, cell migration and invasion, and self-renewal capacity of CSCs and further assessed the expression of CSC marker genes and microRNAs (miRNA) in human pancreatic cancer cells. We found that metformin significantly decreased cell survival, clonogenicity, wound-healing capacity, sphere-forming capacity (pancreatospheres), and increased disintegration of pancreatospheres in both gemcitabine-sensitive and gemcitabine-resistant pancreatic cancer cells. Metformin also decreased the expression of CSC markers,CD44, EpCAM,EZH2, Notch-1, Nanog and Oct4, and caused reexpression of miRNAs (let-7a,let-7b, miR-26a, miR-101, miR-200b, and miR-200c) that are typically lost in pancreatic cancer and especially in pancreatospheres. We also found that reexpression of miR-26a by transfection led to decreased expression of EZH2 and EpCAM in pancreatic cancer cells. These results clearly suggest that the biologic effects of metformin are mediated through reexpression of miRNAs and decreased expression of CSC-specific genes, suggesting that metformin could be useful for overcoming therapeutic resistance of pancreatic cancer cells. Cancer Prev Res; 5(3); 355–64. ©2011 AACR.

Pancreatic cancer: understanding and overcoming chemoresistance

Pancreatic cancer is a highly aggressive malignancy. This feature is believed to be partly attributable to the chemotherapy-resistant characteristics of specific subgroups of pancreatic cancer cells, namely those with an epithelial–mesenchymal transition (EMT) phenotype and cancer stem cells. Accumulating evidence suggests that several new and emerging concepts might be important in the drug-resistant phenotype of these cell types. An understanding of the molecular mechanisms underlying drug resistance in patients with pancreatic cancer might help researchers to devise novel strategies to overcome such resistance. In particular, microRNAs (miRNAs) seem to be critical regulators of drug resistance in pancreatic cancer cells. Selective and targeted elimination of cells with an EMT phenotype and cancer stem cells could be achieved by regulating the expression of specific miRNAs.

Curcumin Analogue CDF Inhibits Pancreatic Tumor Growth by Switching on Suppressor microRNAs and Attenuating EZH2 Expression

The histone methyltransferase EZH2 is a central epigenetic regulator of cell survival, proliferation, and cancer stem cell (CSC) function. EZH2 expression is increased in various human cancers, including highly aggressive pancreatic cancers, but the mechanisms underlying for its biologic effects are not yet well understood. In this study, we probed EZH2 function in pancreatic cancer using diflourinated-curcumin (CDF), a novel analogue of the turmeric spice component curcumin that has antioxidant properties. CDF decreased pancreatic cancer cell survival, clonogenicity, formation of pancreatospheres, invasive cell migration, and CSC function in human pancreatic cancer cells. These effects were associated with decreased expression of EZH2 and increased expression of a panel of tumor-suppressive microRNAs (miRNA), including let-7a, b, c, d, miR-26a, miR-101, miR-146a, andmiR-200b, c that are typically lost in pancreatic cancer. Mechanistic investigations revealed that reexpression of miR-101 was sufficient to limit the expression of EZH2 and the proinvasive cell surface adhesion molecule EpCAM. In an orthotopic xenograft model of human pancreatic cancer, administration of CDF inhibited tumor growth in a manner associated with reduced expression of EZH2, Notch-1, CD44, EpCAM, and Nanog and increased expression of let-7, miR-26a, and miR-101. Taken together, our results indicated that CDF inhibited pancreatic cancer tumor growth and aggressiveness by targeting an EZH2-miRNA regulatory circuit for epigenetically controlled gene expression.

Over‐expression of FoxM1 leads to epithelial–mesenchymal transition and cancer stem cell phenotype in pancreatic cancer cells

FoxM1 is known to play important role in the development and progression of many malignancies including pancreatic cancer. Studies have shown that the acquisition of epithelial‐to‐mesenchymal transition (EMT) phenotype and induction of cancer stem cell (CSC) or cancer stem‐like cell phenotypes are highly inter‐related, and contributes to drug resistance, tumor recurrence, and metastasis. The molecular mechanism(s) by which FoxM1 contributes to the acquisition of EMT phenotype and induction of CSC self‐renewal capacity is poorly understood. Therefore, we established FoxM1 over‐expressing pancreatic cancer (AsPC‐1) cells, which showed increased cell growth, clonogenicity, and cell migration. Moreover, over‐expression of FoxM1 led to the acquisition of EMT phenotype by activation of mesenchymal cell markers, ZEB1, ZEB2, Snail2, E‐cadherin, and vimentin, which is consistent with increased sphere‐forming (pancreatospheres) capacity and expression of CSC surface markers (CD44 and EpCAM). We also found that over‐expression of FoxM1 led to decreased expression of miRNAs (let‐7a, let‐7b, let‐7c, miR‐200b, and miR‐200c); however, re‐expression of miR‐200b inhibited the expression of ZEB1, ZEB2, vimentin as well as FoxM1, and induced the expression of E‐cadherin, leading to the reversal of EMT phenotype. Finally, we found that genistein, a natural chemo‐preventive agent, inhibited cell growth, clonogenicity, cell migration and invasion, EMT phenotype, and formation of pancreatospheres consistent with reduced expression of CD44 and EpCAM. These results suggest, for the first time, that FoxM1 over‐expression is responsible for the acquisition of EMT and CSC phenotype, which is in part mediated through the regulation of miR‐200b and these processes, could be easily attenuated by genistein. J. Cell. Biochem. 112: 2296–2306, 2011.

NF-κB Signaling Pathway and Its Therapeutic Implications in Human Diseases

The nuclear factor-κB (NF-κB) pathway is one of the most important cellular signal transduction pathways involved in both physiologic processes and disease conditions. It plays important roles in the control of immune function, inflammation, stress response, differentiation, apoptosis, and cell survival. Moreover, NF-κB is critically involved in the processes of development and progression of cancers. More importantly, recent studies have shown that NF-κB signaling also plays critical roles in the epithelial-mesenchymal transition (EMT) and cancer stem cells. Therefore, targeting of NF-κB signaling pathway could be a potent strategy for the prevention and/or treatment of human cancers and inflammatory diseases.