ZEB1 Enhances Transendothelial Migration and Represses the Epithelial Phenotype of Prostate Cancer Cells

Abstract

Metastatic colonization involves cancer cell lodgment or adherence in the microvasculature and subsequent migration of those cells across the endothelium into a secondary organ site. To study this process further, we analyzed transendothelial migration of human PC-3 prostate cancer cells in vitro. We isolated a subpopulation of cells, TEM4-18, that crossed an endothelial barrier more efficiently, but surprisingly, were less invasive than parental PC-3 cells in other contexts in vitro. Importantly, TEM4-18 cells were more aggressive than PC-3 cells in a murine metastatic colonization model. Microarray and FACS analysis of these cells showed that the expression of many genes previously associated with leukocyte trafficking and cancer cell extravasation were either unchanged or down-regulated. Instead, TEM4-18 cells exhibited characteristic molecular markers of an epithelial-to-mesenchymal transition (EMT), including frank loss of E-cadherin expression and up-regulation of the E-cadherin repressor ZEB1. Silencing ZEB1 in TEM4-18 cells resulted in increased E-cadherin and reduced transendothelial migration. TEM4-18 cells also express N-cadherin, which was found to be necessary, but not sufficient for increased transendothelial migration. Our results extend the role of EMT in metastasis to transendothelial migration and implicate ZEB1 and N-cadherin in this process in prostate cancer cells. INTRODUCTIONMetastatic prostate cancer is a lethal disease and the second most frequent cause of cancer-related mortality in men in the United States (Jemal et al., 2008). Unfortunately, for such a prevalent disease, much remains unknown about the cellular and molecular mechanisms underlying prostate cancer metastasis. Extravasation, a step within the metastatic cascade, is the process whereby cancer cells exit the circulation via migration through an endothelial monolayer into the parenchyma of a secondary organ site (Wood, 1958). For extravasation to occur, cancer cells, perhaps associated with a thrombus, must first contact the microvascular endothelium, becoming entrapped in small-diameter vessels or adhering specifically to the luminal surface of the endothelium (Warren and Vales, 1972;Kramer and Nicolson, 1979). Some evidence suggests that extravasation is an efficient process, whereas other studies indicate that in some cases it might not occur at all because cancer cells proliferate intraluminally before rupturing microvessels (Crissman et al., 1985;Lapis et al., 1988;Luzzi et al., 1998). Adding to this complexity is that the mechanism of extravasation may depend on the tumor type and vascular bed involved. Thus, as with many other aspects of metastasis, questions remain about the basic mechanisms and the overall role of extravasation in the metastatic cascade.The passage of cancer cells across the endothelium, or transendothelial migration, is thought to be conceptually similar to leukocyte diapedesis (for a recent review see Miles et al., 2008). The extent to which this is true remains controversial, but several classes ...

ZEB1 Coordinately Regulates Laminin-332 and β4 Integrin Expression Altering the Invasive Phenotype of Prostate Cancer Cells*

“…Pro-migratory Laminin-332-Previously we found that although TEM4-18 cells more efficiently crossed an endothelial monolayer and colonized tissues in mice following intravenous injection than the bulk PC-3 population from which they were isolated, they were less invasive than PC-3 cells in other in vitro invasion assays (3). To explore these seemingly inconsistent findings, we hypothesized that PC-3 cells might secrete a matrix-associated component that facilitates cell invasion in Transwell assays.…”

“…Considerable effort over the past decades has defined a number of mechanisms that contribute to the invasive behavior of metastatic cancer cells such as elevated secretion of matrix-degrading enzymes, altered expression of matrix components or their receptors, and increased motility in response to tumor microenvironmental cues. Epithelial-to-mesenchymal transition (EMT) 3 is a mechanism involved in multiple aspects of mammalian development, such as gastrulation and formation of the neural crest, whereby cells lose epithelial identity and gain the ability to move to distant sites in the organism and thus is an attractive paradigm for understanding metastasis (1). Although abundant experimental and some clinical evidence for EMT in cancer exists, the extent to which this mechanism contributes to metastasis remains controversial (2).…”

“…We recently isolated a subpopulation of the PC-3 prostate cancer cell line, TEM4-18, that was proficient in transendothelial migration and displayed hallmarks of EMT (3). We found that TEM4-18 cells differentially express a dual zinc finger homeodomain transcription factor ZEB1 (also known as ZFHX1a, ␦EF1, or TCF-8), which represses a variety of epithelial genes in TEM4-18 cells and has been implicated in EMT in both developmental and pathological contexts, including cancer (for review, see Ref.…”

Pathogenetic Influences of Human Herpesvirus 8 (HHV-8) in Prostate Cancer Progression

“…This ERG-induced epithelial dedifferentiation occurs as the result of ERG activating EZH2-mediated epigenetic modifications (140). Lastly, ERG has been shown to promote EMT and to up-regulate transcription factors that drive EMT, including Zeb1/2 and Snail1/2 (196)(197)(198). The mechanisms by which ERG promotes the development and progression of prostate cancer concern many of the same themes that this project has identified as mechanisms by which HHV-8 promotes an androgenindependent phenotype.…”

“…[187][188][189][190] Herpesvirus -etiological agent for Kaposi's sarcoma (KS), tumor of endothelial and lymphatic origin 101 ERG expression critical for lineage specific differentiation of endothelial cells, angiogenesis and expression of endothelial markers [187][188][189][190] KS lesions composed of "spindle cells," express endothelial and lymphatic markers; virus induces expression of numerous proangiogenic proteins 130 TMPRSS2-ERG gene fusion most prevalent genetic mutation in prostate cancer 27,28 transforms epithelial cells in part by activating MAP kinases 191,192 Virus-encoded proteins activate numerous host cell signaling pathways critical to KS development 130 Oncogenic fusion induces NF-κB activation and inflammation through TLR4 signaling 199 HHV-8 strong inducer of inflammation; activation of NF-κB critical in KS pathology [111][112][113] TMPRSS2-ERG mediates epigenetic regulation through EZH2 140 Viral latency maintained by EZH2 147,148 TMPRSS2-ERG induces epithelialmesenchymal transition [196][197][198] Induces epithelial-mesenchymal transition 149,150 epithelial dedifferentiation and the loss of expression of prostate-defining genes, such as PSA (194,195). This ERG-induced epithelial dedifferentiation occurs as the result of ERG activating EZH2-mediated epigenetic modifications (140).…”