Veronica Henderson

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.

Snail transcription factor negatively regulates maspin tumor suppressor in human prostate cancer cells.

BackgroundMaspin, a putative tumor suppressor that is down-regulated in breast and prostate cancer, has been associated with decreased cell motility. Snail transcription factor is a zinc finger protein that is increased in breast cancer and is associated with increased tumor motility and invasion by induction of epithelial-mesenchymal transition (EMT). We investigated the molecular mechanisms by which Snail increases tumor motility and invasion utilizing prostate cancer cells.MethodsExpression levels were analyzed by RT-PCR and western blot analyses. Cell motility and invasion assays were performed, while Snail regulation and binding to maspin promoter was analyzed by luciferase reporter and chromatin immunoprecipitation (ChIP) assays.ResultsSnail protein expression was higher in different prostate cancer cells lines as compared to normal prostate epithelial cells, which correlated inversely with maspin expression. Snail overexpression in 22Rv1 prostate cancer cells inhibited maspin expression and led to increased migration and invasion. Knockdown of Snail in DU145 and C4-2 cancer cells resulted in up-regulation of maspin expression, concomitant with decreased migration. Transfection of Snail into 22Rv1 or LNCaP cells inhibited maspin promoter activity, while stable knockdown of Snail in C4-2 cells increased promoter activity. ChIP analysis showed that Snail is recruited to the maspin promoter in 22Rv1 cells.ConclusionsOverall, this is the first report showing that Snail can negatively regulate maspin expression by directly repressing maspin promoter activity, leading to increased cell migration and invasion. Therefore, therapeutic targeting of Snail may be useful to re-induce expression of maspin tumor suppressor and prevent prostate cancer tumor progression.

Snail promotes cell migration through PI3K/AKT-dependent Rac1 activation as well as PI3K/AKT-independent pathways during prostate cancer progression

Snail, a zinc-finger transcription factor, induces epithelial-mesenchymal transition (EMT), which is associated with increased cell migration and metastasis in cancer cells. Rac1 is a small G-protein which upon activation results in formation of lamellipodia, the first protrusions formed by migrating cells. We have previously shown that Snail promotes cell migration through down-regulation of maspin tumor suppressor. We hypothesized that Snails regulation of cell migration may also involve Rac1 signaling regulated by PI3K/AKT and/or MAPK pathways. We found that Snail overexpression in LNCaP and 22Rv1 prostate cancer cells increased Rac1 activity associated with increased cell migration, and the Rac1 inhibitor, NSC23766, could inhibit Snail-mediated cell migration. Conversely, Snail downregulation using shRNA in the aggressive C4–2 prostate cancer cells decreased Rac1 activity and cell migration. Moreover, Snail overexpression increased ERK and PI3K/AKT activity in 22Rv1 prostate cancer cells. Treatment of Snail-overexpressing 22Rv1 cells with LY294002, PI3K/AKT inhibitor or U0126, MEK inhibitor, decreased cell migration significantly, but only LY294002 significantly reduced Rac1 activity, suggesting that Snail promotes Rac1 activation via the PI3K/AKT pathway. Furthermore, 22Rv1 cells overexpressing Snail displayed decreased maspin levels, while inhibition of maspin expression in 22Rv1 cells with siRNA, led to increased PI3K/AKT, Rac1 activity and cell migration, without affecting ERK activity, suggesting that maspin is upstream of PI3K/AKT. Overall, we have dissected signaling pathways by which Snail may promote cell migration through MAPK signaling or alternatively through PI3K/AKT-Rac1 signaling that involves Snail inhibition of maspin tumor suppressor. This may contribute to prostate cancer progression.

Targeting the Nuclear Cathepsin L CCAAT Displacement Protein/Cut Homeobox Transcription Factor-Epithelial Mesenchymal Transition Pathway in Prostate and Breast Cancer Cells with the Z-FY-CHO Inhibitor

ABSTRACT The epithelial mesenchymal transition (EMT) promotes tumor migration and invasion by downregulating epithelial markers such as E-cadherin and upregulating mesenchymal markers such as vimentin. Cathepsin L (Cat L) is a cysteine protease that can proteolytically activate CCAAT displacement protein/cut homeobox transcription factor (CUX1). We hypothesized that nuclear Cat L may promote EMT via CUX1 and that this could be antagonized with the Cat L-specific inhibitor Z-FY-CHO. Mesenchymal prostate (ARCaP-M and ARCaP-E overexpressing Snail) and breast (MDA-MB-468, MDA-MB-231, and MCF-7 overexpressing Snail) cancer cells expressed lower E-cadherin activity, higher Snail, vimentin, and Cat L activity, and a p110/p90 active CUX1 form, compared to epithelial prostate (ARCaP-E and ARCaP-Neo) and breast (MCF-7 and MCF-7 Neo) cancer cells. There was increased binding of CUX1 to Snail and the E-cadherin promoter in mesenchymal cells compared to epithelial prostate and breast cells. Treatment of mesenchymal cells with the Cat L inhibitor Z-FY-CHO led to nuclear-to-cytoplasmic relocalization of Cat L, decreased binding of CUX1 to Snail and the E-cadherin promoter, reversed EMT, and decreased cell migration/invasion. Overall, our novel data suggest that a positive feedback loop between Snail-nuclear Cat L-CUX1 drives EMT, which can be antagonized by Z-FY-CHO. Therefore, Z-FY-CHO may be an important therapeutic tool to antagonize EMT and cancer progression.

Snail mediates invasion through uPA/uPAR and the MAPK signaling pathway in prostate cancer cells

Epithelial-mesenchymal transition (EMT) is a process by which cancer cells acquire mesenchymal properties, such as induction of vimentin, while epithelial-associated genes like E-cadherin are lost. This enables cells to be more metastatic. Factors that are able to induce EMT include growth factors such as transforming growth factor-β (TGF-β) and epidermal growth factor, and transcription factors such as Snail. Snail-induced EMT promotes migration and invasion and we hypothesized that this may be mediated by the activity of urokinase-type plasminogen activator (uPA) and its receptor (uPAR). LNCaP, 22Rv1 and ARCaP human prostate cancer (CaP) cells stably transfected with empty vector control (Neo) or constitutively active Snail exhibited increased cell invasion. Superarray analysis revealed an upregulation in uPA and uPAR RNA expression in Snail-transfected ARCaP cells compared with that of a Neo control. In addition, the protein expression levels of Snail, uPA and uPAR were measured by western blot analysis which showed that overexpression of Snail increased uPA and uPAR protein levels. The activity of uPA in conditioned media was measured using an ELISA which revealed that uPA activity was elevated in LNCaP, 22Rv1 and ARCaP cells overexpressing Snail. Additionally, transient silencing of uPAR in ARCaP cells overexpressing Snail using short interfering RNA resulted in abrogation of Snail-mediated invasion. Snail overexpression was associated with increased extracellular-signal-regulated kinase activity, and antagonism of this activity with mitogen-activated protein (MAPK) inhibitor, UO126, inhibited cell invasion and decreased uPA activity. Therefore, Snail-mediated cell invasion in human CaP cells may occur via the regulation of uPA/uPAR and the MAPK signaling pathway.

Abstract C60: Snail transcription factor contributes to bone metastasis in prostate and breast cancer cells

Prostate cancer that is hormone refractory and has metastasized preferentially to bone is the main cause of prostate cancer death, especially in African American men. Receptor activator of nuclear factor kappa B ligand (RANKL) and its receptor (RANK) contributes to bone metastatic lesions and bisphosphonates such as Zometa and Fosamax have been used as antagonists of RANKL for the treatment of breast and prostate cancer metastasis. African American men have the highest bone mineral density compared to any other race, and the role this may play in prostate cancer metastasis to bone is not clear. A better understanding of bone metastasis may lead to alternative treatment options for metastatic prostate cancer. Snail transcription factor is important early in development and in cancer cells and promotes cancer cell migration and progression by inducing epithelial mesenchymal transition (EMT). We have observed increased expression of Snail in prostate cancer bone metastatic human patient samples. We hypothesized that Snail can mediate EMT-mediated prostate cancer migration towards bone of high bone mineral density and mediate the vicious cycle of tumor-tumor microenvironment reciprocal interactions through calcium and RANKL signaling. We generated an EMT model for prostate and breast cancer utilizing the ARCaP human prostate and MCF-7 breast cancer cells overexpressing Snail and identified increased RANKL expression that was associated with increased osteoclastogenesis both in vitro and in vivo, as well as decreased bone volume and density. We utilized pre-molded bone discs which are allograft bone/polyurethane (PUR) composite bone void fillers with tunable properties that have advantage over existing bone implant models in that it contains bigger pore sizes that support rapid cellular infiltration and remodeling. We treated the bone discs with hydrochloric acid which decreased the bone density to a ratio of 1:1.18 for low (HCL-treated): high (untreated) bone density, which is quite close to the 1: 1.2 ratio seen in Caucasian vs African American men. Incubation of these bone discs with prostate or breast cancer cells overexpressing Snail led to increased calcium release from bone of high density as compared to low density. We are currently testing whether this increased calcium release in response to Snail may promote paracrine cell proliferation. Since Snail is not required by adult cells except during injury, targeting Snail that is mainly expressed by cancer cells may antagonize metastatic lesions in bone without affecting normal bone in other areas of the body. Citation Format: Basil A. Smith, Veronica Henderson, Christopher Coke, Jerald Dumas, Cimona Hinton, Manu Platt, Leland K. Chung, Majd Zayzafoon, Valerie A. Odero-Marah. Snail transcription factor contributes to bone metastasis in prostate and breast cancer cells. [abstract]. In: Proceedings of the Sixth AACR Conference: The Science of Cancer Health Disparities; Dec 6–9, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2014;23(11 Suppl):Abstract nr C60. doi:10.1158/1538-7755.DISP13-C60

Association of Epithelial Mesenchymal Transition with prostate and breast health disparities

African Americans (AA) have higher death rates due to prostate and breast cancer as compared to Caucasian Americans (CA), and few biomarkers have been associated with this disparity. In our study we investigated whether epithelial-mesenchymal transition (EMT) with a focus on Snail and Cathepsin L (Cat L), could potentially be two markers associated with prostate and breast health disparities. We have previously shown that Snail can increase Cat L protein and activity in prostate and breast cancer. Western blot and real-time PCR analyses showed that mesenchymal protein expression (Snail, vimentin, Cat L) and Cat L activity (shown by zymography) was higher in AA prostate cancer cells as compared to CA normal transformed RWPE-1 prostate epithelial cells, and androgen-dependent cells, and comparable to metastatic CA cell lines. With respect to breast cancer, mesenchymal markers were higher in TNBC compared to non-TNBC cells. The higher mesenchymal marker expression was functionally associated with higher proliferative and migratory rates. Immunohistochemistry showed that both nuclear Snail and Cat L expression was significantly higher in cancer compared to normal for CA and Bahamas prostate patient tissue. Interestingly, AA normal tissue stained higher for nuclear Snail and Cat L that was not significantly different to cancer tissue for both prostate and breast tissue, but was significantly higher than CA normal tissue. AA TNBC tissue also displayed significantly higher nuclear Snail expression compared to CA TNBC, while no significant differences were observed with Luminal A cancer tissue. Therefore, increased EMT in AA compared to CA that may contribute to the more aggressive disease.

Epithelial-Mesenchymal Transition (EMT) and Prostate Cancer

Typically the normal epithelial cells are a single layer, held tightly by adherent proteins that prevent the mobilization of the cells from the monolayer sheet. During prostate cancer progression, the epithelial cells can undergo epithelial-mesenchymal transition or EMT, characterized by morphological changes in their phenotype from cuboidal to spindle-shaped. This is associated with biochemical changes in which epithelial cell markers such as E-cadherin and occludins are down-regulated, which leads to loss of cell-cell adhesion, while mesenchymal markers such as vimentin and N-cadherin are up-regulated, thereby allowing the cells to migrate or metastasize to different organs. The EMT transition can be regulated directly and indirectly by multiple molecular mechanisms including growth factors and cytokines such as transforming growth factor-beta (TGF-β), epidermal growth factor (EGF) and insulin-like growth factor (IGF), and signaling pathways such as mitogen-activated protein kinase (MAPK) and Phosphatidylinositol 3-Kinase (PI3K). This signaling subsequently induces expression of various transcription factors like Snail, Twist, Zeb1/2, that are also known as master regulators of EMT. Various markers associated with EMT have been reported in prostate cancer patient tissue as well as a possible association with health disparities. There has been consideration to therapeutically target EMT in prostate cancer patients by targeting the EMT signaling pathways.

Snail transcription factor NLS and importin β1 regulate the subcellular localization of Cathepsin L and Cux1

Several recent studies have highlighted an additional unexpected localization and site of action for Cathepsin L (Cat L) protease within the nucleus in breast, colon and prostate cancer, however, its role in the nucleus was unclear. It was proposed to mediate proteolytic processing of the transcription factor CCAAT-displacement protein/cut homeobox transcription factor (Cux1) from the full-length p200 isoform to generate the p110 and p90 isoforms, of which the p110 isoform was shown to act as a cell cycle regulator to accelerate entry into the S phase. The p110 isoform has also been shown to bind to the promoter regions of Snail and E-cadherin to activate Snail and inactivate E-cadherin transcription, thus promoting epithelial mesenchymal transition (EMT). Mechanistic studies on what drives Cat L nuclear localization have not been reported. Our hypothesis is that Snail shuttles into the nucleus with Cat L through binding to importin-β. Snail knockdown with siRNA in MDA-MB-468 breast cancer cells led to nuclear to cytoplasmic shuttling of Cat L and decreased levels of Cux1, while overexpression of Snail in MCF-7 breast cancer cells or HEK-293 human embryonic kidney cells led to increased nuclear expression of both Cat L and Cux1. Additionally, transient transfection of Snail NLS mutants not only abrogated Snail nuclear localization but also nuclear localization of Cat L and Cux1. Interestingly, importin β1 knockdown with siRNA decreased Snail and Cux1 levels, as well as nuclear localization of Cat L. Therefore, we show for the first time that the nuclear localization of Cat L and its substrate Cux1can be positively regulated by Snail NLS and importin β1, suggesting that Snail, Cat L and Cux1 all utilize importin β1 for nuclear import.

Abstract 1692: Role of snail in cancer-bone microenvironment interactions

Prostate Cancer (PCa) is the second leading cause of cancer death in American men and African Americans are twice as likely to get PCa as their Caucasian counterparts. As with most forms of cancer, PCa patients’ mortality is mainly attributed to complications caused by metastasis of the disease to organs critical for survival such as bone. As such, it is important to understand cancer-bone microenvironment interactions in order to develop therapeutics that will slow or halt the process of cancer metastasis. It is also known that African Americans have a higher bone mineral density compared to any other race. Snail1 is a zinc-finger transcription factor that induces epithelial-mesenchymal transition (EMT) which is associated with cell migration and metastasis in cancer cells. In preliminary studies, cancer cells co-cultured with bovine bone discs led to increased calcium release that was higher in cancer cells overexpressing Snail, as well as in bone discs of higher density. We hypothesized that cancer cell-bone interactions would promote higher calcium release from bone by cancer cells overexpressing Snail, which would lead to increased paracrine cell migration. For this study, we utilized prostate (ARCaP) or breast (MCF-7) cancer cells stably overexpressing Snail as well as prostate (C4-2) cancer cells with stable Snail knockdown. Cancer cells were co-cultured with bovine bone disc or Hydroxyapatite (HA; inorganic component of bone) of different densities to represent the African American vs Caucasian bone ratio. The conditioned media was then used for to assay calcium levels, test paracrine cell viability and migration assays using C4-2 parental cells. We observed that calcium levels were elevated in conditioned media from cancer cell-bone co-cultures, compared to media alone or media plus bone, and this could be antagonized by EGTA, a calcium chelator. It was increased with higher bone density as well as with bone co-cultured with MCF-7-Snail, ARCaP-Snail and C4-2 cancer cells as compared to MCF-7 Neo, ARCaP-Neo or C4-2 with Snail knockdown. When we utilized the conditioned media for a paracrine cell viability assay, there was no significant differences. However, C4-2 cancer-bone co-culture conditioned media increased paracrine cell migration which was decreased by Snail knockdown as well as lower bone density. Hence Snail expressing PCa cells co-cultured with HA led to increased paracrine cell migration. We are currently studying the signaling mechanism(s) involved in this cancer-bone interactions. In conclusion, our study shows that Snail can mediate cancer-bone microenvironment interactions that can possibly promote increased cell migration towards bone of high mineral density such as is found in African Americans. GRANT SUPPORT: 1P20MD002285 Citation Format: Veronica M. Henderson, Liza J. Burton, Simone M. Howard, Valerie A. Odero-Marah. Role of snail in cancer-bone microenvironment interactions. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1692.