Liza J. Burton

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.

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.

Muscadine Grape Skin Extract Induces an Unfolded Protein Response-Mediated Autophagy in Prostate Cancer Cells: A TMT-Based Quantitative Proteomic Analysis

Muscadine grape skin extract (MSKE) is derived from muscadine grape (Vitis rotundifolia), a common red grape used to produce red wine. Endoplasmic reticulum (ER) stress activates the unfolded protein response (UPR) that serves as a survival mechanism to relieve ER stress and restore ER homeostasis. However, when persistent, ER stress can alter the cytoprotective functions of the UPR to promote autophagy and cell death. Although MSKE has been documented to induce apoptosis, it has not been linked to ER stress/UPR/autophagy. We hypothesized that MSKE may induce a severe ER stress response-mediated autophagy leading to apoptosis. As a model, we treated C4-2 prostate cancer cells with MSKE and performed a quantitative Tandem Mass Tag Isobaric Labeling proteomic analysis. ER stress response, autophagy and apoptosis were analyzed by western blot, acridine orange and TUNEL/Annexin V staining, respectively. Quantitative proteomics analysis indicated that ER stress response proteins, such as GRP78 were greatly elevated following treatment with MSKE. The up-regulation of pro-apoptotic markers PARP, caspase-12, cleaved caspase-3, -7, BAX and down-regulation of anti-apoptotic marker BCL2 was confirmed by Western blot analysis and apoptosis was visualized by increased TUNEL/Annexin V staining upon MSKE treatment. Moreover, increased acridine orange, and LC3B staining was detected in MSKE-treated cells, suggesting an ER stress/autophagy response. Finally, MSKE-mediated autophagy and apoptosis was antagonized by co-treatment with chloroquine, an autophagy inhibitor. Our results indicate that MSKE can elicit an UPR that can eventually lead to apoptosis in prostate cancer cells.

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.

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.

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 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 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.

Abstract B93: Nuclear cathepsin L (cat L) is associated with epithelial mesenchymal transition (emt) in prostate cancer cells

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. The Epithelial Mesenchymal Transition (EMT) is a key event in tumor migration and invasion processes whereby epithelial cells lose polarity together with cell-cell contacts associated with loss of epithelial markers such as E-cadherin and then undergo a dramatic remodeling of the cytoskeleton associated with expression of mesenchymal markers such as vimentin, as well as increased production of extracellular matrix (ECM)-degrading proteases. Snail promotes EMT by using its zinc finger domains to repress epithelial-associated target genes such as E-cadherin and occludin. Cathepsin L (Cat L) is a cysteine protease involved in cell invasion and has recently been associated with poor prognosis in colon and breast cancer when Cat L is localized in the nucleus. CDP/CUX can be proteolytically activated by Cat L from p200 inactive form to p110 and p90 active forms, and subsequently repress E-cadherin and activate Snail by binding to their promoter regions. We recently published that Snail could up-regulate Cat L expression and activity in prostate cancer cells. We hypothesized that nuclear Cat L may promote EMT and prostate cancer progression and that antagonizing Cat L activity would lead to Mesenchymal Epithelial Transition (MET). We stained African American and Caucasian human normal and prostate cancer tissue from the Fox Chase Cancer Center for Snail and Cat L to examine expression. We utilized prostate cancer cell lines and EMT models to analyze EMT markers and Cat L expression by western blot analysis, Q-PCR and immunofluorescence. We also examined Cat L activity using gelatin zymography and subsequently treated mesenchymal cells with 1 µM, 5 µM or 20 µM Z-FY-CHO (Cat L specific inhibitor) for 72 hours. We also performed invasion and migration assays. Staining of human prostate patient tissue revealed that Snail and Cat L levels were lower in normal tissue vs cancer tissue and nuclear localization of Cat L appeared in earlier stages of prostate cancer in African American tissue compared to that of Caucasian cancer tissue. Interestingly, analysis of epithelial (ARCaP-E) and mesenchymal (ARCaP-M or ARCaP-E overexpressing Snail) prostate cancer cells revealed that mesenchymal cells displaying higher Snail expression showed increased Cat L expression and activity, in addition to Cat L being localized in the cytoplasm and nucleus, whereas Cat L was predominantly cytoplasmic in epithelial cells with low Snail expression. Additionally, the African American cell lines (E006-AA and E006-AA ht) displayed high Snail and Cat L expression/activity compared to LNCaP cells. Mesenchymal cells expressed higher p110 and p90 active form of CDP/CUX than epithelial cells that expressed the p200 inactive form. Additionally, Snail overexpression increased p110/90 CDP/CUX in the nuclear fraction of ARCaP-E overexpressing Snail cells. Treatment of mesenchymal cells with Cat L inhibitor, Z-FY-CHO resulted in decreased vimentin and Cat L expression, while E-cadherin increased, suggestive of MET. This was associated with decreased cell migration and invasion. We further observed that treatment with Z-FY-CHO changes the localization of Cat L from nuclear to cytoplasmic. Overall, our novel data suggests that Snail may promote nuclear Cat L that may be associated with proteolytic degradation of CDP/CUX and EMT, and the inhibition of Cat L could play a role in reverting EMT and preventing tumor progression. Citation Format: Liza J. Burton, Jodi Dougan, Valerie Odero-Marah. Nuclear cathepsin L (cat L) is associated with epithelial mesenchymal transition (emt) in prostate cancer cells. [abstract]. In: Proceedings of the Eighth AACR Conference on The Science of Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; Nov 13-16, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2016;25(3 Suppl):Abstract nr B93.