Eun Jin Seo

TAZ Mediates Lysophosphatidic Acid-Induced Migration and Proliferation of Epithelial Ovarian Cancer Cells

Background: Transcriptional co-activator with PDZ-binding motif (TAZ), a downstream effector of the Hippo pathway, has been reported to regulate organ size, tissue homeostasis, and tumorigenesis by acting as a transcriptional co-activator. Lysophosphatidic acid (LPA) is a bioactive lipid implicated in tumorigenesis and metastasis of ovarian cancer through activation of G protein-coupled receptors. However, the involvement of TAZ in LPA-induced tumorigenesis of ovarian cancer has not been elucidated. Methods: In order to demonstrate the role of TAZ in LPA-stimulated tumorigenesis, the effects of LPA on TAZ expression and cell migration were determined by Western blotting and chemotaxis analyses in R182 human epithelial ovarian cancer cells. Results and Conclusion: Treatment of R182 cells with the LPA receptor inhibitor Ki16425 blocked LPA-induced cell migration. In addition, transfection of R182 cells with small interfering RNA specific for LPA receptor 1 resulted in abrogation of LPA-stimulated cell migration. LPA induced phosphorylation of ERK and p38 MAP kinase in R182 cells and pretreatment of cells with the MEK-ERK pathway inhibitor U0126, but not the p38 MAPK inhibitor SB202190, resulted in abrogation of LPA-induced cell migration. Pretreatment of R182 cells with U0126 attenuated LPA-induced mRNA levels of TAZ and its transcriptional target genes, such as CTGF and CYR61, without affecting phosphorylation level of YAP. These results suggest that MEK-ERK pathway plays a key role in LPA-induced cell migration and mRNA expression of TAZ in R182 cells, without affecting stability of TAZ protein. In addition, small interfering RNA-mediated silencing of TAZ expression attenuated LPA-stimulated migration of R182 cells. These results suggest that TAZ plays a key role in LPA-stimulated migration of epithelial ovarian cancer cells.

FOXP1 functions as an oncogene in promoting cancer stem cell-like characteristics in ovarian cancer cells

Ovarian cancer has the highest mortality rate of all gynecological cancers with a high recurrence rate. It is important to understand the nature of recurring cancer cells to terminally eliminate ovarian cancer. The winged helix transcription factor Forkhead box P1 (FOXP1) has been reported to function as either oncogene or tumor-suppressor in various cancers. In the current study, we show that FOXP1 promotes cancer stem cell-like characteristics in ovarian cancer cells. Knockdown of FOXP1 expression in A2780 or SKOV3 ovarian cancer cells decreased spheroid formation, expression of stemness-related genes and epithelial to mesenchymal transition-related genes, cell migration, and resistance to Paclitaxel or Cisplatin treatment, whereas overexpression of FOXP1 in A2780 or SKOV3 ovarian cancer cells increased spheroid formation, expression of stemness-related genes and epithelial to mesenchymal transition-related genes, cell migration, and resistance to Paclitaxel or Cisplatin treatment. In addition, overexpression of FOXP1 increased promoter activity of ABCG2, OCT4, NANOG, and SOX2, among which the increases in ABCG2, OCT4, and SOX2 promoter activity were dependent on the presence of FOXP1-binding site. In xenotransplantation of A2780 ovarian cancer cells into nude mice, knockdown of FOXP1 expression significantly decreased tumor size. These results strongly suggest FOXP1 functions as an oncogene by promoting cancer stem cell-like characteristics in ovarian cancer cells. Targeting FOXP1 may provide a novel therapeutic opportunity for developing a relapse-free treatment for ovarian cancer patients.

Autotaxin Regulates Maintenance of Ovarian Cancer Stem Cells through Lysophosphatidic Acid‐Mediated Autocrine Mechanism

Ovarian cancer shows high mortality due to development of resistance to chemotherapy and relapse. Cancer stem cells (CSCs) have been suggested to be a major contributor in developing drug resistance and relapse in ovarian cancer. In this study, we isolated CSCs through sphere culture of A2780, SKOV3, OVCAR3 epithelial ovarian cancer cells and primary ovarian cancer cells from patients. We identified heat‐stable factors secreted from ovarian CSCs stimulated migration and proliferation of CSCs. Mass spectrometry and ELISA analysis revealed that lysophosphatidic acid (LPA) was significantly elevated in CSC culture media compared with non‐CSC culture media. Treatment of CSCs with LPA resulted in augmented CSC characteristics such as sphere‐forming ability, resistance to anticancer drugs, tumorigenic potential in xenograft transplantation, and high expression of CSC‐associated genes, including OCT4, SOX2, and aldehyde dehydrogenase 1. Treatment of CSCs with LPA receptor 1‐specific inhibitors or silencing of LPA receptor 1 expression abrogated the LPA‐stimulated CSC properties. Autotaxin, an LPA‐producing enzyme, is highly secreted from ovarian CSCs, and pharmacological inhibition or knockdown of autotaxin markedly attenuated the LPA‐producing, tumorigenic, and drug resistance potentials of CSCs. Clinicopathological analysis showed a significant survival disadvantage of patients with positive staining of autotaxin. In addition, we further identified that AKT1 activity was upregulated in ovarian CSCs through an LPA‐dependent mechanism and silencing of AKT1 expression led to suppression of CSC characteristics. These results suggest that autotaxin‐LPA‐LPA receptor 1‐AKT1 signaling axis is critical for maintaining CSC characteristics through an autocrine loop and provide a novel therapeutic target for ovarian CSCs. Stem Cells 2016;34:551–564

Hypoxia-NOTCH1-SOX2 signaling is important for maintaining cancer stem cells in ovarian cancer

Hypoxia and NOTCH signaling have been reported to be associated with the self-renewal and drug resistance of cancer stem cells (CSCs). However, the molecular mechanisms by which hypoxia and NOTCH signaling stimulate the self-renewal and drug resistance of ovarian CSCs are poorly understood. In the present study, we identified SOX2 as a key transcription factor for CSC-like characteristics in the downstream of hypoxia-induced NOTCH signaling in epithelial ovarian cancer cells. Hypoxic treatment or overexpression of intracellular domain of NOTCH1 (NICD1) in ovarian cancer cells increased sphere formation, drug resistance, and expression of CSC-associated genes such as SOX2, ALDH, and ABC transporters. Hypoxic treatment increased the expression of NICD1, and hypoxic treatment or NICD1 overexpression increased SOX2 promoter activity, which was inhibited by deletion of HIF-1 or CSL binding sites. Furthermore, DAPT treatment decreased the effect of hypoxic treatment, and SOX2 knockdown decreased the effect of hypoxic treatment and NICD overexpression on sphere formation and drug resistance in established ovarian cancer cell lines and primary ovarian cancer cells. These results suggest that hypoxia-NOTCH1-SOX2 signaling axis is important for activation of ovarian CSCs, which may provide a novel opportunity for developing therapeutics to eradicate CSCs in ovarian cancer patients.

Krüppel-like factor 4 mediates lysophosphatidic acid-stimulated migration and proliferation of PC3M prostate cancer cells

Prostate cancer is the most frequently diagnosed malignancy and the second leading cause of cancer mortality among men in the United States. Accumulating evidence suggests that lysophosphatidic acid (LPA) serves as an autocrine/paracrine mediator to affect initiation, progression and metastasis of prostate cancer. In the current study, we demonstrate that LPA stimulates migration and proliferation of highly metastatic human prostate cancer, PC-3M-luc-C6 cells. LPA-induced migration of PC-3M-luc-C6 cells was abrogated by pretreatment of PC-3M-luc-C6 cells with the LPA receptor 1/3 inhibitor Ki16425 or small interfering RNA (siRNA)-mediated silencing of endogenous LPA receptor 1, implicating a key role of the LPA-LPA receptor 1 signaling axis in migration of PC-3M-luc-C6 cells. In addition, LPA treatment resulted in augmented expression levels of Krüppel-like factor 4 (KLF4), and siRNA or short-hairpin RNA (shRNA)-mediated silencing of KLF4 expression resulted in the abolishment of LPA-stimulated migration and proliferation of PC-3M-luc-C6 cells. shRNA-mediated silencing of KLF4 expression resulted in the inhibition of in vivo growth of PC-3M-luc-C6 cells in a xenograft transplantation animal model. Taken together, these results suggest a key role of LPA-induced KLF4 expression in cell migration and proliferation of prostate cancer cells in vitro and in vivo.

Crucial role of HMGA1 in the self-renewal and drug resistance of ovarian cancer stem cells

Cancer stem cells are a subpopulation of cancer cells characterized by self-renewal ability, tumorigenesis and drug resistance. The aim of this study was to investigate the role of HMGA1, a chromatin remodeling factor abundantly expressed in many different cancers, in the regulation of cancer stem cells in ovarian cancer. Spheroid-forming cancer stem cells were isolated from A2780, SKOV3 and PA1 ovarian cancer cells by three-dimensional spheroid culture. Elevated expression of HMGA1 was observed in spheroid cells along with increased expression of stemness-related genes, such as SOX2, KLF4, ALDH, ABCB1 and ABCG2. Furthermore, spheroid A2780 cells, compared with adherent cells, showed higher resistance to chemotherapeutic agents such as paclitaxel and doxorubicin. HMGA1 knockdown in spheroid cells reduced the proliferative advantage and spheroid-forming efficiency of the cells and the expression of stemness-related genes. HMGA1 overexpression in adherent A2780 cells increased cancer stem cell properties, including proliferation, spheroid-forming efficiency and the expression of stemness-related genes. In addition, HMGA1 regulated ABCG2 promoter activity through HMGA1-binding sites. Knockdown of HMGA1 in spheroid cells reduced resistance to chemotherapeutic agents, whereas the overexpression of HMGA1 in adherent ovarian cancer cells increased resistance to chemotherapeutic agents in vitro. Furthermore, HMGA1-overexpressing A2780 cells showed a significant survival advantage after chemotherapeutic agent treatment in a xenograft tumorigenicity assay. Together, our results provide novel insights regarding the critical role of HMGA1 in the regulation of the cancer stem cell characteristics of ovarian cancer cells, thus suggesting that HMGA1 may be an important target in the development of therapeutics for ovarian cancer patients.

Efficient Production of Retroviruses Using PLGA/bPEI-DNA Nanoparticles and Application for Reprogramming Somatic Cells

Reprogramming of somatic cells to pluripotent cells requires the introduction of factors driving fate switches. Viral delivery has been the most efficient method for generation of induced pluripotent stem cells. Transfection, which precedes virus production, is a commonly-used process for delivery of nucleic acids into cells. The aim of this study is to evaluate the efficiency of PLGA/ bPEI nanoparticles in transfection and virus production. Using a modified method of producing PLGA nanoparticles, PLGA/bPEI-DNA nanoparticles were examined for transfection efficiency and virus production yield in comparison with PLGA-DNA, bPEI-DNA nanoparticles or liposome-DNA complexes. After testing various ratios of PLGA, bPEI, and DNA, the ratio of 6:3:1 (PLGA:bPEI:DNA, w/w/w) was determined to be optimal, with acceptable cellular toxicity. PLGA/bPEI-DNA (6:3:1) nanoparticles showed superior transfection efficiency, especially in multiple gene transfection, and viral yield when compared with liposome-DNA complexes. The culture supernatants of HEK293FT cells transfected with PLGA/bPEI-DNA of viral constructs containing reprogramming factors (Oct4, Sox2, Klf4, or c-Myc) successfully and more efficiently generated induced pluripotent stem cell colonies from mouse embryonic fibroblasts. These results strongly suggest that PLGA/bPEI-DNA nanoparticles can provide significant advantages in studying the effect of multiple factor delivery such as in reprogramming or direct conversion of cell fate.