Basil A. Smith

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.

Muscadine grape skin extract can antagonize Snail-cathepsin L-mediated invasion, migration and osteoclastogenesis in prostate and breast cancer cells

To develop new and effective chemopreventive agents against bone metastasis, we assessed the effects of muscadine grape skin extract (MSKE), whose main bioactive component is anthocyanin, on bone turnover, using prostate and breast cancer cell models overexpressing Snail transcription factor. MSKE has been shown previously to promote apoptosis in prostate cancer cells without affecting normal prostate epithelial cells. Snail is overexpressed in prostate and breast cancer, and is associated with increased invasion, migration and bone turnover/osteoclastogenesis. Cathepsin L (CatL) is a cysteine cathepsin protease that is overexpressed in cancer and involved in bone turnover. Snail overexpression in prostate (LNCaP, ARCaP-E) and breast (MCF-7) cancer cells led to increased CatL expression/activity and phosphorylated STAT-3 (pSTAT-3), compared to Neo vector controls, while the reverse was observed in C4-2 (the aggressive subline of LNCaP) cells with Snail knockdown. Moreover, CatL expression was higher in prostate and breast tumor tissue compared to normal tissue. MSKE decreased Snail and pSTAT3 expression, and abrogated Snail-mediated CatL activity, migration and invasion. Additionally, Snail overexpression promoted osteoclastogenesis, which was significantly inhibited by the MSKE as effectively as Z-FY-CHO, a CatL-specific inhibitor, or osteoprotegerin, a receptor activator of nuclear factor kappa B ligand (RANKL) antagonist. Overall, these novel findings suggest that Snail regulation of CatL may occur via STAT-3 signaling and can be antagonized by MSKE, leading to decreased cell invasion, migration and bone turnover. Therefore, inhibition using a natural product such as MSKE could potentially be a promising bioactive compound for bone metastatic cancer.

Camalexin-Induced Apoptosis in Prostate Cancer Cells Involves Alterations of Expression and Activity of Lysosomal Protease Cathepsin D

Camalexin, the phytoalexin produced in the model plant Arabidopsis thaliana, possesses antiproliferative and cancer chemopreventive effects. We have demonstrated that the cytostatic/cytotoxic effects of camalexin on several prostate cancer (PCa) cells are due to oxidative stress. Lysosomes are vulnerable organelles to Reactive Oxygen Species (ROS)-induced injuries, with the potential to initiate and or facilitate apoptosis subsequent to release of proteases such as cathepsin D (CD) into the cytosol. We therefore hypothesized that camalexin reduces cell viability in PCa cells via alterations in expression and activity of CD. Cell viability was evaluated by MTS cell proliferation assay in LNCaP and ARCaP Epithelial (E) cells, and their respective aggressive sublines C4-2 and ARCaP Mesenchymal (M) cells, whereby the more aggressive PCa cells (C4-2 and ARCaPM) displayed greater sensitivity to camalexin treatments than the lesser aggressive cells (LNCaP and ARCaPE). Immunocytochemical analysis revealed CD relocalization from the lysosome to the cytosol subsequent to camalexin treatments, which was associated with increased protein expression of mature CD; p53, a transcriptional activator of CD; BAX, a downstream effector of CD, and cleaved PARP, a hallmark for apoptosis. Therefore, camalexin reduces cell viability via CD and may present as a novel therapeutic agent for treatment of metastatic prostate cancer cells.

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.

The phytoalexin camalexin mediates cytotoxicity towards aggressive prostate cancer cells via reactive oxygen species

Camalexin is a phytoalexin that accumulates in various cruciferous plants upon exposure to environmental stress and plant pathogens. Besides moderate antibacterial and antifungal activity, camalexin was reported to also exhibit antiproliferative and cancer chemopreventive effects in breast cancer and leukemia. We studied the cytotoxic effects of camalexin treatment on prostate cancer cell lines and whether this was mediated by reactive oxygen species (ROS) generation. As models, we utilized LNCaP and its aggressive subline, C4-2, as well as ARCaP cells stably transfected with empty vector (Neo) control or constitutively active Snail cDNA that represents an epithelial to mesenchymal transition (EMT) model and displays increased cell migration and tumorigenicity. We confirmed previous studies showing that C4-2 and ARCaP-Snail cells express more ROS than LNCaP and ARCaP-Neo, respectively. Camalexin increased ROS, decreased cell proliferation, and increased apoptosis more significantly in C4-2 and ARCaP-Snail cells as compared to LNCaP and ARCaP-Neo cells, respectively, while normal prostate epithelial cells (PrEC) were unaffected. Increased caspase-3/7 activity and increased cleaved PARP protein shown by Western blot analysis was suggestive of increased apoptosis. The ROS scavenger N-acetyl cysteine (NAC) antagonized the effects of camalexin, whereas the addition of exogenous hydrogen peroxide potentiated the effects of camalexin, showing that camalexin is mediating its effects through ROS. In conclusion, camalexin is more potent in aggressive prostate cancer cells that express high ROS levels, and this phytoalexin has a strong potential as a novel therapeutic agent for the treatment of especially metastatic prostate cancer.

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

Abstract 542: Camalexin, the phytoalexin from Cruciferous Plants, decreases cell proliferation and induces apoptosis in prostate cancer cells

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Camalexin, the phytoalexin produced in the model plant Arabidopsis thaliana, accumulates in various cruciferous plants upon exposure to environmental stress and plant pathogens. Besides moderate antibacterial and antifungal activity, camalexin was reported to also exhibit antiproliferative and cancer chemopreventive effects. We studied the effects of camalexin treatment on viability of several prostate cancer (PCa) cell lines and whether cytostatic/cytotoxic effects of camalexin-treated prostate cancer cells were due to reactive oxygen species (ROS) generation. As models, we utilized LNCaP and its aggressive subline, C4-2, as well as ARCaP cells stably transfected with empty vector (Neo) control or constitutively active Snail cDNA that represents an EMT model. Previous studies have shown that C4-2 and ARCaP-Snail cells express more ROS than LNCaP and ARCaP-Neo, respectively. We hypothesized that camalexin would decrease cell proliferation and increase apoptosis in PCa cell lines and that the more aggressive cell lines expressing more ROS would be more sensitive to camalexin. Our results showed that camalexin treatments led to higher ROS as shown spectrofluorometrically by the oxidation of H2DCFDA, and this was decreased by co-treatment with NAC. Cell viability using MTS proliferation assay indicated that camalexin treatments decreased cell proliferation more significantly in C4-2 and ARCaP-Snail cells, as compared to LNCaP and ARCaP-Neo cells, respectively, while PrEC normal prostate epithelial cells were unaffected. Conversely, camalexin treatments increased apoptosis and mitocasp activity, which assesses caspase3/7 activity. This was associated with increased p21 and p27 anti-proliferative markers and cleaved PARP apoptotic marker as shown by western blot analysis. NAC reduced camalexin-induced ROS in ARCaP-Snail cells and abrogated camalexin-mediated cell proliferation decrease and apoptosis increase, showing that camalexin is mediating its effects through ROS. In conclusion, camalexin retarded prostate cancer cell proliferation and induced apoptosis via increased ROS generation. Interestingly, it was more potent in aggressive PCa cells expressing higher levels of ROS and did not affect normal cells. Hence, this phytoalexin has a strong potential as a novel therapeutic agent for the treatment of especially metastatic prostate cancer cells that express high levels of ROS. Supported by grants P20MD002285-01 & G12RR003062-22. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 542. doi:1538-7445.AM2012-542

Abstract 3384: The effects of mono- and combination-treatment of phytochemicals and antimicrotubule chemotherapeutic agents on prostate cancer cell proliferation and expression of EMT markers

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL PURPOSE: Treatment of androgen-independent metastatic prostate cancer with chemotherapeutic agent Estramustine showed diminishing success. Epidemiological studies showed an association between reduced cancer risk and dietary intake rich in phytochemicals from fruits and vegetables. We studied the molecular mechanisms through which phytochemicals muscadine grape skin extract (MSKE) and camalexin act individually, or in combination with Estramustine, to affect epithelial-mesenchymal transition (EMT), a process involved in prostate cancer progression. EMT can be induced by transcription factors such as Snail, and is associated with decrease in cell adhesion molecules like E-cadherin, and increase in mesenchymal markers such as vimentin. METHODOLOGY: MSKE, Camalexin, and Estramustine, were used in varying concentrations individually, and in combination, to conduct MTS proliferation assay on C4-2 and ARCaP prostate cancer EMT model overexpressing constitutively active Snail cDNA. EMT markers, E-cadherin and vimentin, were examined by Western Blot Analysis following treatments. RESULTS: MSKE or Camalexin with or without Estramustine significantly decreased cell proliferation of C4-2 cells. Surprisingly, ARCaP-Snail cells were more sensitive to MSKE and Estramustine treatments, and showed significant decrease in viability, as compared to control ARCaP-Neo cells. ARCaP-Snail cells treated with 5µg/ml MSKE showed a reversal in EMT as shown by increased E-cadherin and decreased vimentin levels. CONCLUSION: Both mono and combination treatments retarded prostate cancer cell proliferation and may show promise as a therapeutic agent to abrogate EMT and prostate cancer metastasis in the future. Supported by grants P20MD002285-01 & G12RR003062-22. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3384. doi:10.1158/1538-7445.AM2011-3384

Abstract 1595: HMGA2 induces EMT in prostate cancer cells and may be antagonized by camalexin

Prostate cancer is the most diagnosed cancer in men and the second leading cause of death in the United States. African American men have the highest incidence and mortality rates of prostate cancer compared to any other race. Epithelial-mesenchymal transition (EMT) plays a critical role in cancer progression and metastasis. Mesenchymal cells are migratory, invasive, and more resistant to apoptosis. Reactive Oxygen Species (ROS) has been shown to promote EMT. High mobility group A (HMGA2) is a non-histone protein that is highly expressed during the embryogenesis, whereas the gene expression is very low or absent during adulthood. Recent studies have been reported an overexpression of HMGA2 protein in malignant cancers. Loss of Let-7 miRNA (repressor of HMGA2) has been found to induce EMT via upregulation of HMGA2 in prostate cancer. There has been no link between ROS and HMGA2. We have reported that camalexin, a 3-thizol-2-yl-indole, may be a candidate treatment for aggressive prostate cancer cells by ROS-mediated apoptosis. The study demonstrated that treating the prostate cancer cell with camalexin increased ROS levels which contributed to decreased cell proliferation and increased apoptosis. We hypothesize that HMGA2 may regulate EMT in part by inducing ROS and that camalexin may antagonize HMGA2 signaling. We analysed HMGA2 and EMT marker expression in a panel of prostate cancer cell lines by western blot analysis. We also transiently and stably overexpressed wild-type HMGA2 and mutant HMGA2 (missing Let-7 binding site) in LNCaP cells. We measured ROS levels using DCFDA dye that detects hydrogen peroxide. We treated ARCaP-M (mesenchymal) cells with different concentrations of camalexin to analyse HMGA2 expression. Our results showed that HMGA2 is highly expressed in aggressive prostate cancer cell lines (C4-2, ARCaP, E006AA) as compared to RWPE1 and LNCaP cells. The transient overexpression of HMGA2 in LNCaP cells decreased E-cadherin more markedly than with mutant HMGA2 but did not show any changes in ROS. We will repeat this experiment with stable clones from HMGA2 overexpression. ARCaP-M cells treated with camalexin induced ROS and decreased HMGA2 expression. We are currently performing in vivo studies using ARCaP-M cells injected subcutaneously into nude mice followed by treatment with camalexin after the tumor grows to 50 mm 3 . In conclusion, HMGA2 promotes EMT, even more markedly if its Let-7 suppressor binding site is eliminated, and camalexin may target HMGA2 to decrease prostate cancer progression. GRANT SUPPORT: 1P20MD002285; 8G12MD007590 Citation Format: Ohuod A. hawsawi, Basil Smith, Jodi Dougan, Liza J. Burton, Valerie A. Odero-Marah. HMGA2 induces EMT in prostate cancer cells and may be antagonized by camalexin. [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 1595.

Abstract C57: Snail transcription factor can regulate cathepsin L activity in prostate carcinomas

Prostate cancer (PCa) is the most frequently diagnosed cancer in men, the second leading cause of male cancer deaths in the U.S, and also presents the greatest racial disparity of any cancer in the U.S. The incidence and mortality of PCa is higher in African-American men (AA), compared to other ethnic groups. Snail transcription factor expression is increased in prostate cancer and associated with epithelial-mesenchymal transition (EMT), whereby epithelial cancer cells expressing epithelial markers such as E-cadherin transition into spindle-shaped mesenchymal cells expressing markers such as vimentin. Snail can turn on proteases like MMP-9 to increase degradation of basement membrane and extracellular matrix proteins prior to cancer cell invasion and migration. We have preliminary data showing that muscadine grape skin extract (MSKE), derived from muscadine grape (Vitis rotundifolia), a common red grape used to produce red wine can decrease Snail expression in prostate cancer cells. Cathepsin L is a cysteine cathepsin that is overexpressed in prostate cancer and involved in the repression of E-cadherin, a hallmark of EMT. We investigated whether there could be a link between Snail and Cathepsin L. We hypothesize that the Snail expression may be increased in African American tissue as compared to other races and that Snail can induce the increased activity of Cathepsin L which can be antagonized by MSKE. Initially, we examined the expression of Snail and Cathepsin L in prostate cancer cells by Western blot analysis and Cathepsin L Activity via Zymography. The cell lines utilized in this study are LNCaP, C4-2 (the aggressive subline of LNCaP), mesenchymal ARCaP (ARCaP-M) and epithelial ARCaP (ARCaP-E). Preliminary results suggest that Snail expression and Cathepsin L activity is high in prostate cancer cells compared to normal prostate epithelial cells. We also obtained human prostate cancer tissue from the Bahamas which we are currently staining for Snail and Cathepsin L, to compare to staining from tissue microarrays. We have observed so far that there is both Snail and Cathepsin L expression in the tissue samples. We then utilitized LNCaP and ARCaP-E prostate cancer cells stably transfected with an empty Neo vector (LNCaP Neo; ARCaP Neo) or constituitively active Snail cDNA (LNCaP Snail; ARCaP Snail) that have been shown previously to represent an EMT model, to examine Cathepsin L and Stat-3 activity. We observed that Snail overexpression led to increased Cathepsin L and Stat-3 activity compared to cells with the Neo vector. Additionally ARCaP-E cells overexpresing Snail, C4-2, or ARCaP-M cells treated with MSKE (5ug/mL and 20ug/mL) for a period of 72hrs led to decreased Cathepsin L and Stat-3 activity. Furthermore, MSKE reverted EMT as shown by increased E-cadherein expression, decreased vimentin expression and decrease in cell migration. Taken together, these findings suggest that Snail my increase Cathepsin L activity via the Stat-3 signaling pathway in prostate cancer cells and MSKE could potentially be a promising bioactive compound for human cancer prevention and therapy. Citation Format: Liza Burton, Peri Nappagan, Basil Smith, Manu Platt, Camille Ragin, Valerie Odero-Marah. Snail transcription factor can regulate cathepsin L activity in prostate carcinomas. [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 C57. doi:10.1158/1538-7755.DISP13-C57