Ann-Marie Simeone

Programmed Cell Death 4 inhibits breast cancer cell invasion by increasing Tissue Inhibitor of Metalloproteinases-2 expression

High levels of the cyclooxygenase-2 (COX-2) protein have been associated with invasion and metastasis of breast tumors. Both prostaglandin E2 (PGE2) and interleukin-8 (IL-8) have been shown to mediate the invasive activity of COX-2 in breast cancer cells. Here we expand these studies to determine how COX-2 uses PGE2 and IL-8 to induce breast cancer cell invasion. We demonstrated that PGE2 and IL-8 decreased the expression of the tumor suppressor protein Programmed Cell Death 4 (PDCD4). We hypothesized that suppression of PDCD4 expression is vital to the invasive activity of PGE2 and IL-8. In MCF-7 cells overexpressing PDCD4 (MCF-7/PDCD4), PGE2 and IL-8 failed to induce invasion, in contrast to the parental MCF-7 cells, thus indicating that PDCD4 blocks breast cancer cell invasion. MCF-7/PDCD4 cells produced higher levels of the Tissue Inhibitor of Metalloproteinases-2 (TIMP-2) than the parental cells. Silencing TIMP-2 mRNA in MCF-7/PDCD4 cells reversed the anti-invasive effects of PDCD4, allowing PGE2 and IL-8 to induce the invasion of these cells. Here we report the novel findings that suppression of PDCD4 expression is vital for the invasive activity of COX-2 mediated by PGE2 and IL-8, and that PDCD4 increases TIMP-2 expression to inhibit breast cancer cell invasion.

Cyclooxygenase-2 is essential for HER2/neu to suppress N- (4-hydroxyphenyl)retinamide apoptotic effects in breast cancer cells.

We reported that HER2/neu reduces the sensitivity of breast cancer cells to N-(4-hydroxyphenyl)retinamide (4-HPR) by suppressing nitric oxide production. We show that HER2/neu uses Akt to induce cyclooxygenase-2 (COX-2) expression and that inhibition of Akt or COX-2 increases 4-HPR-induced apoptosis and nitric oxide production. Apoptosis induced by the 4-HPR and COX-2 inhibitor combination, although unaffected by an anti-HER2/neu antibody, was reversed by the COX-2 product prostaglandin E2, indicating that COX-2 is a major mechanism by which HER2/neu suppresses 4-HPR apoptosis in breast cancer cells. Combining 4-HPR with COX-2 inhibitors may be a novel chemopreventive strategy against HER2/neu-overexpressing breast tumors.

TIMP-2 mediates the anti-invasive effects of the nitric oxide-releasing prodrug JS-K in breast cancer cells.

IntroductionTumor invasion and metastasis remain a major cause of mortality in breast cancer patients. High concentrations of nitric oxide (NO) suppress tumor invasion and metastasis in vivo. NO prodrugs generate large amounts of NO upon metabolism by appropriate intracellular enzymes, and therefore could have potential in the prevention and therapy of metastatic breast cancer.MethodsThe present study was designed to determine the effects of the NO-releasing prodrug O2-(2,4-dinitrophenyl) 1- [(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate (JS-K) on breast cancer invasion and the mechanisms involved. MDA-MB-231, MDA-MB-231/F10, and MCF-7/COX-2 were the three breast cancer cell lines tested. NO levels were determined spectrophotometrically using a NO assay kit. Invasion and the expression of matrix metalloproteinases (MMPs) and tissue inhibitor of MMPs were determined using Matrigel invasion assays, an MMP array kit and ELISAs. The activity and expression of extracellular signal-regulated kinase 1/2, p38, and c-Jun N-terminal kinase mitogen-activated protein kinases were determined using western blot analyses.ResultsUnder conditions by which JS-K was not cytotoxic, JS-K significantly decreased (P < 0.05) the invasiveness of breast cancer cells across the Matrigel basement membrane, which was directly correlated with NO production. JS-43-126, a non-NO-releasing analog of JS-K, had no effect on NO levels or invasion. JS-K increased (P < 0.05) TIMP-2 production, and blocking TIMP-2 activity with a neutralizing antibody significantly increased (P < 0.05) the invasive activity of JS-K-treated cells across Matrigel. JS-K decreased p38 activity, whereas the activity and the expression of extracellular signal-regulated kinase 1/2 and c-Jun N-terminal kinase were unaffected.ConclusionWe report the novel findings that JS-K inhibits breast cancer invasion across the Matrigel basement membrane, and NO production is vital for this activity. Upregulation of TIMP-2 production is one mechanism by which JS-K mediates its anti-invasive effects. JS-K and other NO prodrugs may represent an innovative biological approach in the prevention and treatment of metastatic breast cancer.

Leptin utilizes Jun N-terminal kinases to stimulate the invasion of MCF-7 breast cancer cells

In breast tumors, high levels of leptin have been associated with increased incidence of breast cancer metastasis. Breast cancer metastasis is directly associated with breast cancer cell invasion. However, whether leptin could augment breast cancer cell invasion is not known. Here we showed that leptin increased the invasiveness and the matrix metallo-proteinase-2 (MMP-2) activity of the MCF-7 breast cancer cell line. Leptin stimulated the phosphorylation of extracellular signals regulated kinases, signal transducers and activators of transcription 3 and Jun N-terminal kinases (JNK); however, only inhibition of JNK decreased leptin-mediated activation of MMP-2. Furthermore, inhibition of JNK suppressed leptin-mediated breast cancer cell invasion. Here we report the novel findings that leptin increased invasion of breast cancer cells by activating JNK, resulting in increased MMP-2 activity.

JS-K, a nitric oxide-releasing prodrug, induces breast cancer cell death while sparing normal mammary epithelial cells

Targeted therapy with reduced side effects is a major goal in cancer research. We investigated the effects of JS-K, a nitric oxide (NO) prodrug designed to release high levels of NO when suitably activated, on human breast cancer cell lines, on non-transformed human MCF-10A mammary cells, and on normal human mammary epithelial cells (HMECs). Cell viability assay, flow cytometry, electron microscopy, and Western blot analysis were used to study the effects of JS-K on breast cancer and on mammary epithelial cells. After a 3-day incubation, the IC50s of JS-K against the breast cancer cells ranged from 0.8 to 3 µM. However, JS-K decreased the viability of the MCF-10A cells by only 20% at 10-µM concentration, and HMECs were unaffected by 10 µM JS-K. Flow cytometry indicated that JS-K increased the percentages of breast cancer cells under-going apoptosis. Interestingly, flow cytometry indicated that JS-K increased acidic vesicle organelle formation in breast cancer cells, suggesting that JS-K induced autophagy in breast cancer cells. Electron microscopy confirmed that JS-K-treated breast cancer cells underwent autophagic cell death. Western blot analysis showed that JS-K induced the expression of microtubule light chain 3-II, another autophagy marker, in breast cancer cells. However, JS-K did not induce apoptosis or autophagy in normal human mammary epithelial cells. These data indicate that JS-K selectively induces programmed cell death in breast cancer cells while sparing normal mammary epithelial cells under the same conditions. The selective anti-tumor activity of JS-K warrants its further investigation in breast tumors.

HER2/neu reduces the apoptotic effects of N-(4-hydroxyphenyl)retinamide (4-HPR) in breast cancer cells by decreasing nitric oxide production

The retinoid N-(4-hydroxyphenyl)retinamide (4-HPR also known as fenretinide) is a potent inducer of apoptosis in breast cancer cells. We observed a 4.5-fold reduction in 4-HPR-mediated apoptosis in MCF-7 breast cancer cells transfected with HER2/neu (MCF-7/HER2) as compared with the parental MCF-7 (MCF-7/WT) cells. Blocking HER2/neu with trastuzumab (HerceptinTM) led to a six-fold increase in 4-HPR-induced apoptosis in HER2/neu-overexpressing cells. These data indicate that HER2/neu reduces the sensitivity of breast cancer cells to 4-HPR. We showed previously that nitric oxide (NO) is essential for 4-HPR to induce apoptosis in breast cancer cells. The inhibitory effects of the 4-HPR and trastuzumab combination correlated with the amount of NO produced in HER2/neu-overexpressing cells. When a NO synthase (NOS) inhibitor was used to block NO production, decreased apoptosis by the 4-HPR and trastuzumab combination was observed. Furthermore, 4-HPR-mediated NOSII expression was lower in MCF-7/HER2 than MCF-7/WT cells, but was increased by trastuzumab in HER2/neu-overexpressing cells. Here we report the novel findings that HER2/neu reduces the ability of 4-HPR to induce apoptosis in breast cancer cells, and that one mechanism by which HER2/neu increases the resistance of breast cancer cells to 4-HPR is by decreasing NOSII-mediated NO production.

Cyclosporin A enhances the apoptotic effects of N-(4-hydroxyphenyl)retinamide in breast cancer cells

4HPR, an analogue of ATRA, effectively induces growth inhibition and apoptosis in breast cancer cell lines and animal models but is ineffective against advanced human breast tumors. Different compounds, including tamoxifen, are currently being tested to increase 4HPR efficacy in the clinic. Here, we report that cyclosporin A selectively increases the ability of 4HPR, but not ATRA, to induce growth inhibition and apoptosis in ER+ and ER− breast cancer cell lines. Increased apoptosis by the 4HPR and cyclosporin A combination was correlated with increased production of the free radical nitric oxide. Thus, the 4HPR and cyclosporin A combination may potentially be a novel therapeutic modality against breast tumors.

Abstract 1426: Cyclooxygenase-2 (COX-2) utilizes Jun N-terminal kinases to induce invasion but not tamoxifen resistance in MCF-7 breast cancer cells

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Elevated Cyclooxygenase-2 (COX-2) expression in breast tumors had been associated with a lower survival rate in patients with Estrogen Receptor α (ERα)-positive tumors. We propose that COX-2 reduces survival rate in breast cancer patients with ERα-positive tumors because COX-2 decreases tamoxifen sensitivity and increases invasive activity of breast cancer cells. We demonstrated that transfection of the COX-2 gene in the tamoxifen-sensitive, ERα-positive MCF-7 breast cancer cell line decreased the cells’ sensitivity to tamoxifen by about 5-fold, and increased their invasive activity by about 3-fold. Furthermore, we demonstrated that COX-2 utilizes PGE2 to activate Protein Kinase C (PKC) to induce these tumorigeneic effects. However, which downstream factors are recruited by the COX-2/PGE2/PKC pathway to induce tamoxifen resistance and breast cancer cell invasion are not known. Here we report that COX-2 increases the phosphorylation of Jun N-terminal kinase (JNK), but not that of extracellular signal regulated kinases (ERK1,2), p38 mitogen activated protein kinase, or Akt. Inhibition of JNK by a chemical inhibitor or siRNA led to decreased COX-2-mediated invasion. Yet, JNK inhibition did not affect COX-2-mediated tamoxifen resistance. We propose that JNK is a promising target to block invasion in patients whose tumors are ERα-positive and present high levels of COX-2. 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 1426. doi:10.1158/1538-7445.AM2011-1426

Role of the prostaglandin receptor EP3 in regulating vasculogenic mimicry of inflammatory breast cancer.

CTRC-AACR San Antonio Breast Cancer Symposium: 2008 Abstracts Abstract #2127 Background: Inflammatory breast cancer is an aggressively angiogenic, metastatic and lethal variant of breast cancer. One of the characteristics of aggressive tumors types such as IBC is the ability of tumor cells to undergo vasculogenic mimicry (VM), which is the formation of organized capillary like structures in vitro and in vivo much like endothelial cells, providing a conduit for oxygen and nutrients to reach hypoxic and nutrient deprived tumor cells that have outgrown their adjacent vascular supply. Using genomic and proteomic profiling as well as Western blotting, immunohistochemistry, invasion assays using the modified Boyden chamber assay as well as digital imaging, we evaluated the signal transduction pathways involved in regulating proliferation, invasion, and VM by IBC tumor cells. Results: We found that cyclooxygenase-2 (Cox-2), which produces the bioactive lipid, prostaglandin E2 (PGE2) is one gene that is highly expressed IBC tumor cell lines, SUM149 and SUM190. The biological activity of PGE2 occurs via binding of PGE2 to one of 4 members of a G protein coupled receptor family, designated as the EP receptors, EP1, EP2, EP3 and EP4. Western blot analysis and immunochemistry revealed that EP3 receptor protein is produced at very high levels by both IBC tumor cell lines to a significantly (p<0.05) greater extent than either human MCF-7 or MDA-MB-231 non-IBC breast tumor cells. Since EP3 is known to be an inhibitory prostanoid receptor, we evaluated the dose dependent effects of the EP3 agonist, sulprostone, on proliferation, invasion, and VM. Sulprostone [0.1, 1, 10 and 100 μm] inhibited proliferation of SUM149 and SUM190 IBC cells in a dose and time dependent manner to a significantly greater extent (p<0.01) than MCF-7 or MDA-MB-231 breast tumor cells. Sulprostone inhibited invasion of SUM149 IBC tumor cells, with no effect on invasion of a basement membrane by the MDA-MB-231 human non-IBC breast tumor cells. SUM149 IBC tumor cells undergo VM, which was completely inhibited by 10 μM sulprostone at 24 hrs, as visualized by Periodic Acid Schiff (PAS) staining and digital imaging. The ability of sulprostone to completely inhibit VM occurred in conjunction with inhibition of production of laminin 5-γ2 fragments and matrix metalloproteinase-2 (MMP-2) activity. Studies are currently underway to determine the in vivo effect of sulprostone on IBC tumor growth, VM, and metastasis of SUM149 IBC xenografts. Discussion: These results suggest that targeting EP3 may provide a selective advantage for abrogating not only proliferation, and invasion but also vasculogenic mimicry that is a key component of the rapid angiogenic program observed in IBC. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 2127.