bay-11-7082 and Breast-Neoplasms

A major clinical problem in the treatment of breast cancer is mortality due to metastasis. Understanding the molecular mechanisms associated with metastasis should aid in designing new therapeutic approaches for breast cancer. Trastuzumab is the main therapeutic option for HER2+ breast cancer patients; however, the molecular basis for trastuzumab resistance (TZR) and subsequent metastasis is not known. Earlier, we found expression of basal-like molecular markers in TZR tissues from patients with invasive breast cancer.(( 1 )) The basal-like phenotype is a particularly aggressive form of breast cancer. This observation suggests that TZR might contribute to an aggressive phenotype. To understand if resistance to TZR can lead to basal-like phenotype, we generated a trastuzumab-resistant human breast cancer cell line (BT-474-R) that maintained human epidermal growth factor receptor 2 (HER2) overexpression and HER2 mediated signaling. Analysis showed that nuclear factor-kappa B (NF-κB) was constitutively activated in the BT-474-R cells, a feature similar to the basal-like tumor phenotype. Pharmacologic inhibition of NF-κB improved sensitivity of BT-474-R cells to trastuzumab. Interestingly, activation of HER2 independent NF-κB is not shown in luminal B breast cancer cells. Our study suggests that by activating the NF-κB pathway, luminal B cells may acquire a HER2+ basal-like phenotype in which NF-κB is constitutively activated; this notion is consistent with the recently proposed "progression through grade" or "evolution of resistance" hypothesis. Furthermore, we identified IKK-α/IKK-β and nuclear accumulation of RelA/p65 as the major determinants in the resistant cells. Thus our study additionally suggests that the nuclear accumulation of p65 may be a useful marker for identifying metastasis-initiating tumor cells and targeting RelA/p65 may limit metastasis of breast and other cancers associated with NF-κB activation.

Topics: Antineoplastic Agents; Breast Neoplasms; Cell Line, Tumor; Drug Resistance, Neoplasm; Female; Humans; I-kappa B Kinase; NF-kappa B; Nitriles; Receptor, ErbB-2; Sulfones; Transcription Factor RelA; Trastuzumab

Although drug resistance is often observed in metastatic recurrence of breast cancer, little is known about the intrinsic drug resistance in such metastases. In the present study, we found, for the first time, that MDA-MB-231BR, a brain metastatic variant of a human breast cancer cell line, was refractory to treatment with 5-fluorouracil (5-FU) even without chronic drug exposure, compared to its parent cell line, MDA-MB-231, and a bone metastatic variant, MDA-MB-231SCP2. Both the mRNA and protein levels of COX-2 and BCL2A1 in MDA-MB-231BR were significantly higher than those in MDA-MB-231 or MDA-MB-231SCP2. Neither the COX-2 inhibitor celecoxib nor the NF-κB inhibitor BAY11-7082 could sensitize MDA-MB-231BR to 5-FU, indicating that COX-2 plays little, if any, role in the resistance of MDA-MB-231BR to 5-FU. Although BCL2-family inhibitor ABT-263 failed to sensitize MDA-MB-231BR to 5-FU at a dose at which ABT-263 is considered to bind to BCL2, BCL2-xL, and BCL2-w, but not to BCL2A1, ABT-263 did sensitize MDA-MB-231BR to 5-FU to a level comparable to that in MDA-MB-231 at a dose of 5 μM, at which ABT-263 may disrupt intracellular BCL2A1 protein interactions. More importantly, BCL2A1 siRNA sensitized MDA-MB-231BR to 5-FU, whereas the overexpression of BCL2A1 conferred 5-FU-resistance on MDA-MB-231. These results indicate that BCL2A1 is a key contributor to the intrinsic 5-FU-resistance in MDA-MB-231BR. It is interesting to note that the drug sensitivity of MDA-MB-231BR was distinct from that of MDA-MB-231SCP2 even though they have the same origin (MDA-MB-231). Further investigations pertinent to the present findings may provide valuable insight into the breast cancer brain metastasis.

Topics: Aniline Compounds; Brain Neoplasms; Breast Neoplasms; Celecoxib; Cell Line, Tumor; Cell Survival; Cyclooxygenase 2; Cyclooxygenase 2 Inhibitors; Drug Resistance, Neoplasm; Female; Fluorouracil; Gene Expression; Humans; Minor Histocompatibility Antigens; NF-kappa B; Nitriles; Protein Interaction Maps; Proto-Oncogene Proteins c-bcl-2; RNA Interference; RNA, Messenger; RNA, Small Interfering; Sulfonamides; Sulfones

Many cancers spread through lymphatic routes, and mechanistic insights of tumour intravasation into the lymphatic vasculature and targets for intervention are limited. The major emphasis of research focuses currently on the molecular biology of tumour cells, while still little is known regarding the contribution of lymphatics.. Breast cancer cell spheroids attached to lymphendothelial cell (LEC) monolayers were used to investigate the process of intravasation by measuring the areas of 'circular chemorepellent-induced defects' (CCID), which can be considered as entry gates for bulky tumour intravasation. Aspects of tumour cell intravasation were furthermore studied by adhesion assay, and siRNA-mediated knockdown of intracellular adhesion molecule-1 (ICAM-1). Replacing cancer spheroids with the CCID-triggering compound 12(S)-hydroxyeicosatetraenoic acid (HETE) facilitated western blot analyses of Bay11-7082- and baicalein-treated LECs.. Binding of LECs to MCF-7 spheroids, which is a prerequisite for CCID formation, was mediated by ICAM-1 expression, and this depended on NF-κB and correlated with the expression of the prometastatic factor S100A4. Simultaneous inhibition of NF-κB with Bay11-7082 and of arachidonate lipoxygenase (ALOX)-15 with baicalein prevented CCID formation additively.. Two mechanisms contribute to CCID formation: ALOX15 via the generation of 12(S)-HETE by MCF-7 cells, which induces directional migration of LECs, and ICAM-1 in LECs under control of NF-κB, which facilitates adhesion of MCF-7 cells to LECs.

Topics: Blotting, Western; Breast Neoplasms; Cell Adhesion; Cell Movement; Chemotaxis; Endothelium, Lymphatic; Female; Humans; Intercellular Adhesion Molecule-1; NF-kappa B; Nitriles; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Spheroids, Cellular; Sulfones; Tumor Cells, Cultured

Photodynamic therapy (PDT) employs photoexcitation of a sensitizer to generate tumor-eradicating reactive oxygen species. We recently showed that irradiating breast cancer COH-BR1 cells after treating with 5-aminolevulinic acid (ALA, a pro-sensitizer) resulted in rapid upregulation of inducible nitric oxide (NO) synthase (iNOS). Apoptotic cell killing was strongly enhanced by an iNOS inhibitor (1400W), iNOS knockdown (kd), or a NO scavenger, suggesting that NO was acting cytoprotectively. Stress signaling associated with these effects was examined in this study. ALA/light-stressed COH-BR1 cells, and also breast adenocarcinoma MDA-MB-231 cells, mounted an iNOS/NO-dependent resistance to apoptosis that proved to be cGMP-independent. Immunocytochemistry and subcellular Western analysis of photostressed COH-BR1 cells revealed a cytosol-to-nucleus translocation of NF-κB which was negated by the NF-κB activation inhibitor Bay11. Bay11 also enhanced apoptosis and prevented iNOS induction, consistent with NF-κB involvement in the latter. JNK and p38 MAP kinase inhibitors suppressed apoptosis, implicating these kinases in death signaling. Post-irradiation extent and duration of JNK and p38 phosphorylation were dramatically elevated by 1400 W or iNOS-kd, suggesting that these activations were suppressed by NO. Regarding pro-survival stress signaling, rapid activation of Akt was unaffected by 1400 W, but prevented by Wortmannin, which also enhanced apoptosis. Thus, a link between upstream Akt activation and iNOS induction was apparent. Furthermore, p53 protein expression under photostress was elevated by iNOS-kd, whereas robust Survivin induction was abolished, consistent with p53 and Survivin being negatively and positively regulated by NO, respectively. Collectively, these findings enhance our understanding of cytoprotective signaling associated with photostress-induced NO and suggest iNOS inhibitor-based approaches for improving PDT efficacy.

Topics: Adenocarcinoma; Aminolevulinic Acid; Apoptosis; Apoptosis Regulatory Proteins; Breast Neoplasms; Cell Line, Tumor; Cytoprotection; Enzyme Inhibitors; Female; Humans; Inhibitor of Apoptosis Proteins; JNK Mitogen-Activated Protein Kinases; MAP Kinase Signaling System; NF-kappa B; Nitric Oxide; Nitric Oxide Synthase Type II; Nitriles; Oxidative Stress; p38 Mitogen-Activated Protein Kinases; Photochemotherapy; Protein Transport; Proto-Oncogene Proteins c-akt; Reactive Oxygen Species; RNA Interference; RNA, Small Interfering; Sulfones; Survivin

Both IκB kinase (IKK) complex and oncgenic protein Myc play important roles in cancer progression, including cancer cell invasiveness and metastasis. The levels of Myc is regulated by the phosphorylation of Myc at Thr58 and Ser62.. In this study, we show that the expression of Myc is associated with IKKα and IKKβ in breast cancers and that Myc is an IKKs substrate. Suppression of IKK activity by either chemical inhibitor or transfection of kinase-dead mutants decreases the phosphorylation of Myc at Ser62 and enhances the degradation of Myc. Consequently, these treatments decrease the tumorigenic and invasive ability of breast cancer cells. Furthermore, doxorubicin, a frequently used anticancer drug in breast cancer, activates IKKs and Myc, thereby increasing invasiveness and tumorigenesis of breast carcinoma MCF7 cells. Inhibition of IKKs prevents these doxorubicin-induced effects.. Our study indicates that IKKs tightly regulate Myc expression through prolonging protein stability, and suggests that IKKs are potentially therapeutic targets and that suppression of IKKs may be used following chemotherapy to reduce the risk of treatment-induced tumor progression.

Topics: Antineoplastic Agents; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Cell Transformation, Neoplastic; Disease Progression; Doxorubicin; Female; Humans; I-kappa B Kinase; NF-kappa B; Nitriles; Protein Binding; Protein Stability; Proto-Oncogene Proteins c-myc; RNA Stability; RNA, Messenger; Signal Transduction; Sulfones

The intravasation of breast cancer into the lymphendothelium is an early step of metastasis. Little is known about the mechanisms of bulky cancer invasion into lymph ducts.. To particularly address this issue, we developed a 3-dimensional co-culture model involving MCF-7 breast cancer cell spheroids and telomerase-immortalised human lymphendothelial cell (LEC) monolayers, which resembles intravasation in vivo and correlated the malignant phenotype with specific protein expression of LECs.. We show that tumour spheroids generate 'circular chemorepellent-induced defects' (CCID) in LEC monolayers through retraction of LECs, which was induced by 12(S)-hydroxyeicosatetraenoic acid (HETE) secreted by MCF-7 spheroids. This 12(S)-HETE-regulated retraction of LECs during intravasation particularly allowed us to investigate the key regulators involved in the motility and plasticity of LECs. In all, 12(S)-HETE induced pro-metastatic protein expression patterns and showed NF-κB-dependent up-regulation of the mesenchymal marker protein S100A4 and of transcriptional repressor ZEB1 concomittant with down-regulation of the endothelial adherence junction component VE-cadherin. This was in accordance with ∼50% attenuation of CCID formation by treatment of cells with 10 μM Bay11-7082. Notably, 12(S)-HETE-induced VE-cadherin repression was regulated by either NF-κB or by ZEB1 since ZEB1 siRNA knockdown abrogated not only 12(S)-HETE-mediated VE-cadherin repression but inhibited VE-cadherin expression in general.. These data suggest an endothelial to mesenchymal transition-like process of LECs, which induces single cell motility during endothelial transmigration of breast carcinoma cells. In conclusion, this study demonstrates that the 12(S)-HETE-induced intravasation of MCF-7 spheroids through LECs require an NF-κB-dependent process of LECs triggering the disintegration of cell-cell contacts, migration, and the generation of CCID.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Antineoplastic Agents; Breast Neoplasms; Carcinoma; Cell Line, Transformed; Cell Movement; Cell Transdifferentiation; Coculture Techniques; Endothelial Cells; Female; Humans; Mesoderm; Neoplasm Invasiveness; NF-kappa B; Nitriles; Signal Transduction; Sulfones; Tumor Cells, Cultured

ErbB2 (HER2, neu) is a receptor tyrosine kinase overexpressed in about 25% of invasive breast carcinomas. Neutrophil gelatinase-associated lipocalin (NGAL) is a secreted glycoprotein expressed in a variety of cancers, including breast carcinomas. NGAL can inhibit erythroid cell production, leading to anemia. Anemia usually occurs in cancer patients and negatively affects quality of life. However, current treatment for cancer-related anemia has potential complications. ErbB2, NGAL, and anemia have all been associated with increased metastasis and poor prognosis in breast cancer patients, although the relationship between ErbB2 and NGAL expression is not clear. Here, using breast cancer cell lines in vitro and transgenic mice carrying the activated c-neu oncogene driven by a mouse mammary tumor virus (MMTV-neu) in vivo, we show that ErbB2 overexpression leads to NGAL upregulation, which is dependent on nuclear factor-kappaB (NF-kappaB) activity. MMTV-neu transgenic mice developed anemia after tumor onset, and anemia progression could be partially arrested by a NF-kappaB inhibitor and ErbB2-targeted therapy. Taken together, upregulation of NGAL by ErbB2 through NF-kappaB activation is involved in cancer-related anemia, and the ErbB2, NF-kappaB, and NGAL pathways may serve as potential therapeutic targets for cancer-related anemia.

Topics: Acute-Phase Proteins; Anemia; Animals; Antineoplastic Agents; Breast Neoplasms; Cell Line, Tumor; Female; Humans; Lipocalin-2; Lipocalins; Mammary Neoplasms, Experimental; Mice; Mice, Transgenic; NF-kappa B; Nitriles; Proto-Oncogene Proteins; Receptor, ErbB-2; RNA, Messenger; Signal Transduction; Sulfones; Up-Regulation

Nuclear factor-kappa B (NF-kappa B) is a known survival pathway, and it may explain differential sensitivity to tumor necrosis factor-alpha (TNF-alpha) and chemotherapeutic-induced apoptosis in apoptotically sensitive (APO+) and apoptotically resistant (APO-) Michigan Cancer Foundation-7 breast cancer cells.. Crystal violet viability and luciferase reporter gene assays were used to determine the inhibitory concentration of viability at 50% (IC(50)) and the inhibitory concentration of activity at 50% (EC(50)) values in APO- and APO+ cells with the selective NF-kappa B inhibitor, BAY 11-7082 (BAY). The apoptotic reporter assay was used to determine the effects of the transfection of the inhibitory kappa B-dominant negative (I kappa B-DN) construct in conjunction with TNF, paclitaxel, or doxorubicin treatments in these cells.. The concentrations at which 50% of cell viability is inhibited (IC(50)) and at which 50% of NF-kappa B activity is inhibited (EC(50)) for BAY in APO- and APO+ cells were 95.24 micromol/L and 1.53 micromol/L, respectively, and 7.62 micromol/L and 2.64 micromol/L, respectively. The IC(50) and the EC(50) values were equivalent for the APO+ cells (P =.665), but not for the APO- cells (P =.025). I kappa B-DN--transfection alone, or with TNF, doxorubicin, or paclitaxel treatments resulted in cell death of both APO- and APO+ cells as compared with vector-control; however, greater cytotoxicity was seen in the APO+ cells. Direct comparison of the APO+ cells versus the APO- cells revealed that these differences were significant (P =.05).. Pharmacologic or molecular inhibition of the NF-kappa B pathway blocked cell survival in MCF-7 APO+ cells, while only molecular inhibition induced cytotoxicity in the APO- cells. Selective manipulation of the NF-kappa B pathway in combination with standard chemotherapeutic agents may lead to an increased potency and efficacy of these agents.

Topics: Antineoplastic Agents; Antineoplastic Agents, Phytogenic; Apoptosis; Breast Neoplasms; Doxorubicin; Drug Resistance, Neoplasm; Female; Gene Expression; Genes, Reporter; Humans; Inhibitory Concentration 50; Luciferases; NF-kappa B; Nitriles; Organic Chemicals; Paclitaxel; Sulfones; Tumor Cells, Cultured