Valerie Odero-Marah

Insulin-like Growth Factor-I–Dependent Up-regulation of ZEB1 Drives Epithelial-to-Mesenchymal Transition in Human Prostate Cancer Cells

The epithelial-to-mesenchymal transition (EMT) is crucial for the migration and invasion of many epithelial tumors, including prostate cancer. Although it is known that ZEB1 overexpression promotes EMT primarily through down-regulation of E-cadherin in a variety of cancers, the soluble ligands responsible for the activation of ZEB1 have yet to be identified. In the present study, we investigated the role of insulin-like growth factor-I (IGF-I) in the regulation of ZEB1 during EMT associated with prostate tumor cell migration. We found that ZEB1 is expressed in highly aggressive prostate cancer cells and that its expression correlates directly with Gleason grade in human prostate tumors (P < 0.001). IGF-I up-regulates ZEB1 expression in prostate cancer cells exhibiting an epithelial phenotype. In prostate cancer cells displaying a mesenchymal phenotype, ZEB1 inhibition reverses the suppression of E-cadherin protein and down-regulates the expression of the mesenchymal markers N-cadherin and fibronectin. Furthermore, ZEB1 blockade decreases migratory and invasive potential in ARCaP(M) compared with the control. These results identify ZEB1 as a key transcriptional regulator of EMT in prostate cancer and suggest that the aberrant expression of ZEB1 in prostate cancer cells occurs in part in response to IGF-I stimulation.

Receptor activator of NF-κB Ligand (RANKL) expression is associated with epithelial to mesenchymal transition in human prostate cancer cells

Epithelial-mesenchymal transition (EMT) in cancer describes the phenotypic and behavioral changes of cancer cells from indolent to virulent forms with increased migratory, invasive and metastatic potential. EMT can be induced by soluble proteins like transforming growth factor β1 (TGFβ1) and transcription factors including Snail and Slug. We utilized the ARCaPE/ARCaPM prostate cancer progression model and LNCaP clones stably overexpressing Snail to identify novel markers associated with EMT. Compared to ARCaPE cells, the highly tumorigenic mesenchymal ARCaPM and ARCaPM1 variant cells displayed a higher incidence of bone metastasis after intracardiac administration in SCID mice. ARCaPM and ARCaPM1 expressed mesenchymal stromal markers of vimentin and N-cadherin in addition to elevated levels of Receptor Activator of NF-κB Ligand (RANKL). We observed that both epidermal growth factor (EGF) plus TGFβ1 treatment and Snail overexpression induced EMT in ARCaPE and LNCaP cells, and EMT was associated with increased expression of RANKL protein. Finally, we determined that the RANKL protein was functionally active, promoting osteoclastogenesis in vitro. Our results indicate that RANKL is a novel marker for EMT during prostate cancer progression. RANKL may function as a link between EMT, bone turnover, and prostate cancer skeletal metastasis.

Maspin Regulates Different Signaling Pathways for Motility and Adhesion in Aggressive Breast Cancer Cells

Previous studies from our laboratory and others have demonstrated that treatment of breast cancer cells with exogenous maspin led to a significant decrease in cell motility, and an increase in cell adhesion to human fibronectin. However, the signaling mechanisms by which maspin, a putative tumor suppressor gene, might regulate cell motility and adhesion have not been previously addressed. In this study, we hypothesized that maspin could inhibit cell motility through the Rho GTPase pathway, specifically by affecting Rac activity. To test this intriguing hypothesis we utilized an experimental approach where invasive and metastatic MDA-MB-231 breast cancer cells were either treated exogenously with recombinant maspin protein, or stably transfected with maspin. The data revealed decreased Rac1 activity within 4 h, and a decrease in the Rac1 effector, PAK1, within 12 h. In addition, an increase in PI3K and ERK1/2 activities within 1 h of recombinant maspin (rMaspin) treatment was observed, which returned to baseline level after 12 h. ERK activity was shown to be downstream of PI3K, as pretreatment with the PI3K inhibitor, LY294002, inhibited the stimulation of ERK activity by rMaspin. Furthermore, rMaspin-treated cells displayed approximately a 30% increase in cell adhesion which was abrogated by pretreatment with LY294002. Increased focal adhesions and stress fibers were observed after 12 h of rMaspin treatment, when the cells were least motile and had reverted to a more epithelial-like phenotype. These data suggest that maspin may inhibit cell motility by regulating Rac1 and subsequently PAK1 activity, and promote cell adhesion via PI3K/ERK pathways. This study provides new insights into the diverse signaling pathways affected by maspin to suppress the metastatic phenotype, and could contribute to novel therapeutic approaches for the treatment of invasive and metastatic breast cancer.

Snail Transcription Factor Regulates Neuroendocrine Differentiation in LNCaP Prostate Cancer Cells

Snail transcription factor induces epithelial–mesenchymal transition (EMT) via decreased cell adhesion‐associated molecules like E‐cadherin, and increased mesenchymal markers like vimentin. We previously established Snail‐mediated EMT model utilizing androgen‐dependent LNCaP cells. These cells express increased vimentin protein and relocalization of E‐cadherin from the cell membrane to the cytosol. Interestingly, Snail transfection in LNCaP cells resulted in cells acquiring a neuroendocrine (NE)‐like morphology with long neurite‐like processes.

The role of Snail in prostate cancer

Prostate cancer is the second most frequently diagnosed cancer and the sixth leading cause of death from cancer in men. Epithelial-mesenchymal transition (EMT) is a process by which cancer cells invade and migrate, and is characterized by loss of cell-cell adhesion molecules such as E-cadherin and increased expression of mesenchymal proteins such as vimentin; EMT is also associated with resistance to therapy. Snail, a master regulator of EMT, has been extensively studied and reported in cancers such as breast and colon; however, its role in prostate cancer is not as widely reported. The purpose of this review is to put together recent facts that summarize Snail signaling in human prostate cancer. Snail is overexpressed in prostate cancer and its expression and activity is controlled via phosphorylation and growth factor signaling. Snail is involved in its canonical role of inducing EMT in prostate cancer cells; however, it plays a role in non-canonical pathways that do not involve EMT such regulation of bone turnover and neuroendocrine differentiation. Thus, studies indicate that Snail signaling contributes to prostate cancer progression and metastasis and therapeutic targeting of Snail in prostate cancer holds promise in „future.

Reciprocal regulation of ZEB1 and AR in triple negative breast cancer cells

Zinc-finger enhancer binding protein (ZEB1) is a transcription factor involved in the progression of cancer primarily through promoting epithelial to mesenchymal transition (EMT). ZEB1 represses the expression of E-cadherin by binding to E-box sequences in the promoter, thus decreasing epithelial differentiation. We show that ZEB1 and androgen receptor (AR) cross-talk in triple negative breast cancer cell lines. Chromatin immunoprecipitation analysis demonstrates that ZEB1 binds directly to the E-box located in the AR promoter. ZEB1 suppression by stably transfecting shRNA in a triple negative breast cancer cell line resulted in a decrease of AR mRNA, protein, and AR downstream targets. ZEB1 knockdown in triple negative breast cancer cells sensitized the cells to bicalutamide by reducing migration compared to the control cells. Conversely, blockade of AR signaling with bicalutamide resulted in a suppression of ZEB1 protein expression in two triple negative breast cancer cell lines. Furthermore, using a breast cancer tissue microarray, a majority of triple negative breast cancers exhibit positive staining for both ZEB1 and AR. Taken together, these results indicate that ZEB1 and AR regulate each other to promote cell migration in triple negative breast cancer cells.

Snail Promotes Epithelial Mesenchymal Transition in Breast Cancer Cells in Part via Activation of Nuclear ERK2

Snail transcription factor is up-regulated in several cancers and associated with increased tumor migration and invasion via induction of epithelial-to-mesenchymal transition (EMT). MAPK (ERK1/2) signaling regulates cellular processes including cell motility, adhesion, and invasion. We investigated the regulation of ERK1/2 by Snail in breast cancer cells. ERK1/2 activity (p-ERK) was higher in breast cancer patient tissue as compared to normal tissue. Snail and p-ERK were increased in several breast cancer cell lines as compared to normal mammary epithelial cells. Snail knockdown in MDA-MB-231 and T47-D breast cancer cells decreased or re-localized p-ERK from the nuclear compartment to the cytoplasm. Snail overexpression in MCF-7 breast cancer cells induced EMT, increased cell migration, decreased cell adhesion and also increased tumorigenicity. Snail induced nuclear translocation of p-ERK, and the activation of its subcellular downstream effector, Elk-1. Inhibiting MAPK activity with UO126 or knockdown of ERK2 isoform with siRNA in MCF-7 Snail cells reverted EMT induced by Snail as shown by decreased Snail and vimentin expression, decreased cell migration and increased cell adhesion. Overall, our data suggest that ERK2 isoform activation by Snail in aggressive breast cancer cells leads to EMT associated with increased cell migration and decreased cell adhesion. This regulation is enhanced by positive feedback regulation of Snail by ERK2. Therefore, therapeutic targeting of ERK2 isoform may be beneficial for breast cancer.

Loss of PTEN permits CXCR4-mediated tumorigenesis through ERK1/2 in prostate cancer cells.

Loss of PTEN is frequently observed in androgen-independent prostate cancer, resulting in the deregulation of metastatic events. SDF1α activation of CXCR4 induces signaling pathways that have been implicated in prostate metastasis and progression to an advanced disease. The pathways of CXCR4 and PTEN converge, leading to the promotion and regulation of tumorigenesis, respectively. However, loss of PTEN may permit CXCR4 to progress prostate cancer to an advanced disease. In the present study, we investigated the involvement of PTEN in CXCR4-mediated tumorigenesis. When screening advanced metastatic prostate cancer cell lines for PTEN, we observed a loss of expression in PC3 and LNCaP cells whereas Du145 expressed wild-type PTEN. All three cell lines were positive for surface expression of CXCR4. Reconsitution of PTEN induced a mesenchymal to epithelial like morphologic change and inhibited CXCR4-mediated migration and proliferation in PC3 cells. Downregulation of PTEN by siRNA enhanced the CXCR4-mediated migratory behavior of Du145 cells. By Western blot analysis, we observed that PTEN inhibited basal AKT phosphorylation but not ERK1/2 phosphorylation in PTEN-expressing cells. Upon CXCR4 stimulation, PTEN inhibited ERK1/2 phosphorylation but not phosphorylation of AKT. The CXCR4-mediated migration of PC3 cells was through the ERK1/2 pathway, as confirmed by chemical inhibitors. On the basis of these studies, we suggest that loss of PTEN permits CXCR4-mediated functions in prostate cancer cells through the ERK1/2 pathway. Antagonizing CXCR4 and downstream signaling cascades may provide an efficient approach for treating patients with advanced prostate cancer when hormone therapy fails to the stop the growth and containment of tumors. Mol Cancer Res; 9(1); 90–102 ©2010 AACR.

Snail negatively regulates cell adhesion to extracellular matrix and integrin expression via the MAPK pathway in prostate cancer cells.

Snail transcription factor induces epithelial-mesenchymal transition (EMT) in which the epithelial cells downregulate cell-cell adhesion genes such as E-Cadherin and upregulate mesenchymal genes such as vimentin, leading to increased invasion and migration. Very little is known about the role of Snail in cellular adhesion to the extracellular matrix. We hypothesized that Snail will lead to decreased cellular adhesion to fibronectin and collagen I matrix through integrin regulation, concomitant with increased cell migration. Androgen-independent C4-2 cells, an aggressive subline of androgen-dependent LNCaP cells, exhibited decreased cell adhesion and increased cell migration on fibronectin and collagen I as compared to LNCaP cells, which was reversed by Snail knock down in C4-2 cells. ARCaP and LNCaP prostate cancer cells stably transfected with Snail displayed decreased adhesion and increased cell migration on fibronectin and collagen I as compared to control Neo-transfected cells, which was reversed by Snail knockdown. Flow cytometry analysis revealed a decrease in a5, a2 and b1 integrin expression in ARCaP Snail-transfected cells that was reversed in Snail knock down cells. We also observed an increase in ERK phosphorylation in ARCaP Snail-transfected cells as compared to control ARCaP-Neo cells, and inhibition of the MAPK pathway with UO126 inhibitor in ARCaP Snail-transfected cells abrogated Snail-mediated decrease in cell adhesion and reinduced a5, a2 and b1 integrin expression. Collectively, these studies define a new role for Snail transcription factor in cell adhesion to the ECM, which may be mediated by MAPK signaling, and may be crucial for cell detachment and subsequent metastasis.

Snail-mediated regulation of reactive oxygen species in ARCaP human prostate cancer cells.

Reactive oxygen species increases in various diseases including cancer and has been associated with induction of epithelial-mesenchymal transition (EMT), as evidenced by decrease in cell adhesion-associated molecules like E-cadherin, and increase in mesenchymal markers like vimentin. We investigated the molecular mechanisms by which Snail transcription factor, an inducer of EMT, promotes tumor aggressiveness utilizing ARCaP prostate cancer cell line. An EMT model created by Snail overexpression in ARCaP cells was associated with decreased E-cadherin and increased vimentin. Moreover, Snail-expressing cells displayed increased concentration of reactive oxygen species (ROS), specifically, superoxide and hydrogen peroxide, in vitro and in vivo. Real Time PCR profiling demonstrated increased expression of oxidative stress-responsive genes, such as aldehyde oxidase I, in response to Snail. The ROS scavenger, N-acetyl cysteine partially reversed Snail-mediated EMT after 7 days characterized by increased E-cadherin levels and decreased ERK activity, while treatment with the MEK inhibitor, UO126, resulted in a more marked effect by 3 days, characterized by cells returning back to the epithelial morphology and increased E-cadherin. In conclusion, this study shows for the first time that Snail transcription factor can regulate oxidative stress enzymes and increase ROS-mediated EMT regulated in part by ERK activation. Therefore, Snail may be an attractive molecule for therapeutic targeting to prevent tumor progression in human prostate cancer.