Peri Nagappan

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.

Inhibitor of Differentiation 4 (ID4) Inactivation Promotes De Novo Steroidogenesis and Castration-Resistant Prostate Cancer

Prostate cancer (PCa) is the most commonly diagnosed cancer in men in the Western world. The transition of androgen-dependent PCa to castration-resistant (CRPC) is a major clinical manifestation during disease progression and presents a therapeutic challenge. Our studies have shown that genetic ablation of inhibitor of differentiation 4 (Id4), a dominant-negative helix loop helix protein, in mice results in prostatic intraepithelial neoplasia lesions and decreased Nkx3.1 expression without the loss of androgen receptor (Ar) expression. ID4 is also epigenetically silenced in the majority of PCa. However, the clinical relevance and molecular pathways altered by ID4 inactivation in PCa are not known. This study investigates the effect of loss of ID4 in PCa cell lines on tumorigenicity and addresses the underlying mechanism. Stable silencing of ID4 in LNCaP cells (L-ID4) resulted in increased proliferation, migration, invasion, and anchorage-independent growth. An increase in the rate of tumor growth, weight, and volume was observed in L-ID4 xenografts compared with that in the LNCaP cells transfected with nonspecific short hairpin RNA (L+ns) in noncastrated mice. Interestingly, tumors were also observed in castrated mice, suggesting that loss of ID4 promotes CRPC. RNA sequence analysis revealed a gene signature mimicking that of constitutively active AR in L-ID4, which was consistent with gain of de novo steroidogenesis. Prostate-specific antigen expression as a result of persistent AR activation was observed in L-ID4 cells but not in L+ns cells. The results demonstrate that ID4 acts as a tumor suppressor in PCa, and its loss, frequently observed in PCa, promotes CRPC through constitutive AR activation.

Cholesterol and phytosterols differentially regulate the expression of caveolin 1 and a downstream prostate cell growth-suppressor gene.

BACKGROUND The purpose of our study was to show the distinction between the apoptotic and anti-proliferative signaling of phytosterols and cholesterol-enrichment in prostate cancer cell lines, mediated by the differential transcription of caveolin-1, and N-myc downstream-regulated gene 1 (NDRG1), a pro-apoptotic androgen-regulated tumor suppressor. METHODS PC-3 and DU145 cells were treated with sterols (cholesterol and phytosterols) for 72h, followed by trypan blue dye-exclusion measurement of necrosis and cell growth measured with a Coulter counter. Sterol induction of cell growth-suppressor gene expression was evaluated by mRNA transcription using RT-PCR, while cell cycle analysis was performed by FACS analysis. Altered expression of Ndrg1 protein was confirmed by Western blot analysis. Apoptosis was evaluated by real time RT-PCR amplification of P53, Bcl-2 gene and its related pro- and anti-apoptotic family members. RESULTS Physiological doses (16microM) of cholesterol and phytosterols were not cytotoxic in these cells. Cholesterol-enrichment promoted cell growth (P<0.05), while phytosterols significantly induced growth-suppression (P<0.05) and apoptosis. Cell cycle analysis showed that contrary to cholesterol, phytosterols decreased mitotic subpopulations. We demonstrated for the first time that cholesterols concertedly attenuated the expression of caveolin-1 (cav-1) and NDRG1 genes in both prostate cancer cell lines. Phytosterols had the opposite effect by inducing overexpression of cav-1, a known mediator of androgen-dependent signals that presumably control cell growth or apoptosis. CONCLUSIONS Cholesterol and phytosterol treatment differentially regulated the growth of prostate cancer cells and the expression of p53 and cav-1, a gene that regulates androgen-regulated signals. These sterols also differentially regulated cell cycle arrest, downstream pro-apoptotic androgen-regulated tumor suppressor, NDRG1 suggesting that cav-1 may mediate pro-apoptotic NDRG1 signals. Elucidation of the mechanism for sterol modulation of growth and apoptosis signaling may reveal potential targets for cancer prevention and/or chemotherapeutic intervention. Sterol regulation of NDRG1 transcription suggests its potential as biomarker for prediction of neoplasms that would be responsive to chemoprevention by phytosterols.

Selectively self-assembling graphene nanoribbons with shaped iron oxide nanoparticles

Nanoflower and nanoplate-shaped iron oxide nanoparticles (FeNPs) were attached onto graphene nanoribbons (GNRs). Raman spectra of FeNP–GNRs reveal red-shifted G-bands along with greatly reduced D-band intensity, indicating a surface-smooth and electron-doped assembly on GNR. Distinctive decorative patterns are associated with unique sizes, shapes, and crystallinities of FeNPs. FeNP-nanoflowers are predominately attached around the edges of the GNRs; whereas multilayer GNRs are sandwich-stacked with FeNP-nanoplates. With the attachment of IgG antibodies, FeNP–GNRs–IgG depict distinguishable aggregation features, which have potential advantages as biosensors to target tumor cells with over-expressed folic acid.

Inactivation of ID4 promotes a CRPC phenotype with constitutive AR activation through FKBP52

Castration‐resistant prostate cancer (CRPC) is the emergence of prostate cancer cells that have adapted to the androgen‐depleted environment of the prostate. In recent years, targeting multiple chaperones and co‐chaperones (e.g., Hsp27, FKBP52) that promote androgen receptor (AR) signaling and/or novel AR regulatory mechanisms have emerged as promising alternative treatments for CRPC. We have shown that inactivation of inhibitor of differentiation 4 (ID4), a dominant‐negative helix loop helix protein, promotes de novo steroidogenesis and CRPC with a gene expression signature that resembles constitutive AR activity in castrated mice. In this study, we investigated the underlying mechanism through which loss of ID4 potentiates AR signaling. Proteomic analysis between prostate cancer cell line LNCaP (L+ns) and LNCaP lacking ID4 (L(−)ID4) revealed elevated levels of Hsp27 and FKBP52, suggesting a role for these AR‐associated co‐chaperones in promoting constitutively active AR signaling in L(−)ID4 cells. Interestingly, protein interaction studies demonstrated a direct interaction between ID4 and the 52‐kDa FK506‐binding protein (FKBP52) in vitro, but not with AR. An increase in FKBP52‐dependent AR transcriptional activity was observed in L(−)ID4 cells. Moreover, pharmacological inhibition of FKBP52‐AR signaling, by treatment with MJC13, attenuated the tumor growth, weight, and volume in L(−)ID4 xenografts. Together, our results demonstrate that ID4 selectively regulates AR activity through direct interaction with FKBP52, and its loss, promotes CRPC through FKBP52‐mediated AR signaling.

Abstract 1131: Snail- and ERK2-dependent signaling enhances breast cancer cell resistance to hydroxytamoxifen

Snail transcription factor and MAPK/ERK signaling regulate EMT and chemotherapy resistance in various tumor models by binding to target promoters (i.e., E-cadherin, maspin, ER-α). ERK1 is expressed during embryogenesis and in non-metastatic cells; ERK2 is implicated during vasculogenesis and promotes stem cell phenotype in triple negative breast cancer. Nuclear-localized ERK is associated with more active and potentially metastatic breast and ovarian carcinoma cells; cytoplasmic-localized ERK is a good prognostic factor. The role that Snail plays during the transition from cytoplasmic ERK1 to nuclear ERK2 has not been investigated. We hypothesized that both nuclear Snail and ERK2 are required for breast cancer cells to become resistant to 4-hydroxytamoxifen (4-OHT). We utilized MCF-7 breast adenocarcinoma cells, MCF-7 transfected stably with empty Neo vector control (MCF-7 Neo) or constitutively active Snail cDNA (MCF-7 Snail). MCF-7 Neo and MCF-7 Snail have previously represented a breast cancer EMT model; we used this model to analyze ERK signaling and therapy response. Preliminary data suggest that MCF-7 Neo express higher levels of p-ERK, ER-α, and E-cadherin and no Snail. MCF-7 Snail expressed higher levels of Snail and vimentin, low levels of p-ERK, and low ER-α and no E-cadherin. MCF-7 Neo expressed higher ERK1 and MCF-7 Snail expressed higher ERK2. Immunocytochemistry indicated that p-ERK was nuclear and cytoplasmic in MCF-7 Neo, but mostly nuclear in MCF-7 Snail. We treated MCF-7 Neo and MCF-7 Snail with control siRNA, ERK1 siRNA, or ERK2 siRNA, then determined the expression of Snail, p-ERK1/2, ERK1/2, E-cadherin, and ER-α. We found that ERK1 siRNA decreased E-cadherin, while ERK2 siRNA increased E-cadherin in MCF-7 Neo. ERK2 siRNA decreased Snail, but ERK1 siRNA had no effect on Snail. ERK1 siRNA increased active ERK2 and ERK2 siRNA increased ERK1 in MCF-7 Neo and MCF-7 Snail. To determine the role that Snail plays in resistance, we treated MCF-7 Neo and MCF-7 Snail with 4-OHT and/or UO126 then mitochondrial permeability and caspase-3/7 activity assays. MCF-7 Neo had higher mitochondrial permeability than MCF-7 Snail, indicative of the dimmer staining using TMRM stain, and MCF-7 Neo had higher caspase-3/7 activity after treatments. Overall, cell death in MCF-7 Snail was caspase-independent. These data imply that Snail and ERK2 collaborate through an unknown mechanism to promote EMT and therapy resistance in breast cancer cells. Targeting Snail and/or ERK2 in advanced breast cancer may be a potential therapeutic or preventive strategy. Citation Format: Bethany N. Smith, Peri Nagappan, Latonia Taliaferro-Smith, Roman Mezencev, Clayton Yates, Cimona Hinton, Valerie Odero-Marah. Snail- and ERK2-dependent signaling enhances breast cancer cell resistance to hydroxytamoxifen. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1131. doi:10.1158/1538-7445.AM2014-1131

Abstract 1485: Snail activation of MAPK pathway may contributes to tumor insensitivity to tamoxifen chemotherapy in breast cancer cells.

Snail is a zinc finger transcriptional repressor that induces epithelial-mesenchymal transitions (EMT) and is expressed at high levels in tumors. Snail contributes to chemotherapy resistance in ovarian, liver, colorectal, and breast carcinomas by activating the MAPK/ERK1/2 (ERK1/2) pathway . ERK1/2 controls cell proliferation, survival, migration, and adhesion. However, a role for MAPK in Snail-mediated EMT in breast cancer is unclear. We hypothesized that Snail promotes EMT by decreasing cell adhesion and increasing cell migration through the MAPK pathway, resulting inresistance to Tamoxifen chemotherapy. Preliminary results suggest that breast cancer cells express variable levels of Snail that is higher than in normal MCF10A cells. We utilized MCF-7 breast adenocarcinoma cells transfected stably with empty Neo vector control (MCF-7 Neo) or constitutively active Snail cDNA (MCF-7 Snail). MCF-7 Neo and MCF-7 Snail have previously represented a breast cancer EMT model; we used this model to examine cell adhesion and cell migration. We injected these cells into female nude mice to examine tumor size. We also examined activation of MAPK pathway using phospo-ERK (p-ERK) antibodies in response to Snail overexpression and the effect of inhibiting this pathway with UO126 (MEK inhibitor, 20uM). Finally, we tested how Snail overexpression affects response to UO126 and/or Tamoxifen treatments using MTS cell proliferation assay. Our results showed that Snail overexpression led to decreased cell adhesion and increased cell migration on collagen and fibronectin in vitro, and increased tumorigenicity in vivo. Interestingly, immunofluorescent analysis revealed that activated ERK1/2 (p-ERK) had cytoplasmic localization in MCF-7 Neo, and exclusive nuclear localization in MCF-7 Snail. Inhibition of activated ERK1/2 with UO126 antagonized cell adhesion primarily in MCF-7 Neo cells, while it decreased cell migration primarily in MCF-7 Snail cells. This would suggest that cytoplasmic p-ERK may be important for cell adhesion while nuclear p-ERK may be important for cell migration. MCF-7 Neo proliferation was consistently higher than MCF-7 Snail indicating that Snail may not play a significant role in cell proliferation, but other EMT functions like migration and adhesion. Tamoxifen treatment did not affect MCF-7 Snail cells morphologically, but led to sickly-appearing MCF-7 Neo cells. Additionally, MCF-7 Snail cells treated with Tamoxifen proliferated at similar rates to control untreated cells but co-treatment with UO126 greatly inhibited cell proliferation. This suggests that antagonizing nuclear p-ERK may sensitize breast cancer cells to Tamoxifen therapy. In conclusion, our study shows that Snail activation of MAPK pathway specifically within the nucleus promotes resistance to Tamoxifen and future ERK1/2 inhibition within the nucleus may sensitize cells to chemotherapy. Citation Format: Bethany N. Smith, Peri Nagappan, Mahandranauth Chetram, LaTonia Taliaferro-Smith, Clayton Yates, Cimona Hinton, Valerie Odero-Marah. Snail activation of MAPK pathway may contributes to tumor insensitivity to tamoxifen chemotherapy in breast cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1485. doi:10.1158/1538-7445.AM2013-1485

Abstract 5747: Elucidating the mechanisms by which Snail transcription factor promotes resistance to chemotherapy in breast carcinomas

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Snail (Snail1) is a zinc finger transcription factor that can induce EMT and is expressed at high levels in tumors. Maspin is a potential tumor suppressor, whose expression is lost as breast and prostate tumors progress during EMT. Maspin can be transcriptionally repressed by androgen receptor (AR) in prostate cancer cells. We hypothesized that overexpression of Snail will inhibit maspin expression and confer resistance to chemotherapy. Initially, we examined the expression of Snail and maspin in a panel of breast epithelial and breast cancer cells by RT-PCR and Western blot analysis. Preliminary results suggest that breast cancer cells express variable levels of Snail that is higher in breast cancer cells than in the normal MCF10A cells. We then utilized MCF-7 cells transfected stably with an empty Neo vector (MCF-7 Neo) or constitutively active Snail cDNA (MCF-7 Snail) that have been shown previously to represent an EMT model, and examined maspin, estrogen receptor alpha (ER-α) and AR levels by western blot analysis. MCF-7 Snail cells had upregulated levels of Snail and lower levels of maspin, ER-α and AR, as compared to MCF-7 Neo. MCF-7 Neo and MCF-7 Snail cells were also xenografted into nude mice and tissue sections stained immunohistochemically for Snail, maspin, and ER-α. Tumor volumes of MCF-7 Snail tumor xenografts were significantly larger than the MCF-7 Neo xenografts. MCF-7 Snail tissue sections expressed higher levels of Snail and lower levels of maspin and ER-α as compared to MCF-7 Neo sections. MCF-7 Neo and MCF-7 Snail cells were also treated with estradiol (E2, 0.01uM), tamoxifen (TAM, 10uM), and docetaxel (DOX, 1uM) individually; and in combination with E2 plus TAM and TAM plus DOX for periods of 24h and 72h to analyze changes in cell morphology and EMT marker expression. No effect on cell morphology or cell death was observed in MCF-7 Snail cells after TAM treatments, whereas TAM induced cell death in MCF-7 Neo. Expression of Snail was increased with E2 treatments and decreased with TAM, DOX, and combination treatments in MCF-7 Neo and MCF-7 Snail. AR expression decreased further in MCF-7 Snail cells and MCF-7 Neo following treatments with TAM and DOX. Overall, the Snail overexpression appears to promote resistance to TAM and DOX chemotherapeutic agents. The mechanism of Snail-regulated EMT needs to be investigated further to determine changes in breast cancer progression. 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 5747. doi:1538-7445.AM2012-5747