anisomycin and Breast-Neoplasms

Topics: Animals; Anisomycin; Antineoplastic Agents; Aspergillus; Breast Neoplasms; Cell Culture Techniques; Cell Proliferation; Cell Survival; Drug Screening Assays, Antitumor; Female; Humans; MCF-7 Cells; Porifera

MALAT1 (metastasis associated lung adenocarcinoma transcript1) is a conserved long non-coding RNA, known to regulate gene expression by modulating transcription and post-transcriptional pre-mRNA processing of a large number of genes. MALAT1 expression is deregulated in various tumors, including breast cancer. However, the significance of such abnormal expression is yet to be fully understood. In this study, we demonstrate that regulation of aggressive breast cancer cell traits by MALAT1 is not predicted solely based on an elevated expression level but is context specific. By performing loss- and gain-of-function studies, both under in vitro and in vivo conditions, we demonstrate that MALAT1 facilitates cell proliferation, tumor progression and metastasis of triple-negative breast cancer (TNBC) cells despite having a comparatively lower expression level than ER or HER2-positive breast cancer cells. Furthermore, MALAT1 regulates the expression of several cancer metastasis-related genes, but displays molecular subtype specific correlations with such genes. Assessment of the prognostic significance of MALAT1 in human breast cancer (n=1992) revealed elevated MALAT1 expression was associated with decreased disease-specific survival in ER negative, lymph node negative patients of the HER2 and TNBC molecular subtypes. Multivariable analysis confirmed MALAT1 to have independent prognostic significance in the TNBC lymph node negative patient subset (HR=2.64, 95%CI 1.35- 5.16, p=0.005). We propose that the functional significance of MALAT1 as a metastasis driver and its potential use as a prognostic marker is most promising for those patients diagnosed with ER negative, lymph node negative breast cancer who might otherwise mistakenly be stratified to have low recurrence risk.

Topics: Anisomycin; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Cell Proliferation; Epithelial-Mesenchymal Transition; Estrogen Receptor alpha; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Lymphatic Metastasis; Multivariate Analysis; Neoplasm Metastasis; Neoplasm Recurrence, Local; Phenotype; Prognosis; Risk; RNA Splicing; RNA, Long Noncoding; Triple Negative Breast Neoplasms

Anisomycin was identified in a screen of clinical compounds as a drug that kills breast cancer cells (MDA16 cells, derived from the triple negative breast cancer cell line, MDA-MB-468) that express high levels of an efflux pump, ABCB1. We show the MDA16 cells died by a caspase-independent mechanism, while MDA-MB-468 cells died by apoptosis. There was no correlation between cell death and either protein synthesis or JNK activation, which had previously been implicated in anisomycin-induced cell death. In addition, anisomycin analogues that did not inhibit protein synthesis or activate JNK retained the ability to induce cell death. These data suggest that either a ribosome-ANS complex is a death signal in the absence of JNK activation or ANS kills cells by binding to an as yet unidentified target.

Topics: Anisomycin; Apoptosis; Breast Neoplasms; Cell Line, Tumor; Enzyme Activation; Humans; MAP Kinase Kinase 4; Protein Biosynthesis; Protein Synthesis Inhibitors

Transcription factor ATF-2 is a nuclear target of stress-activated protein kinases, such as p38, which are activated by various extracellular stresses, including UV light. Here, we show that ATF-2 plays a critical role in hypoxia- and high-cell-density-induced apoptosis and the development of mammary tumors. Compared to wild-type cells, Atf-2(-/-) mouse embryonic fibroblasts (MEFs) were more resistant to hypoxia- and anisomycin-induced apoptosis but remained equally susceptible to other stresses, including UV. Atf-2(-/-) and Atf-2(+/-) MEFs could not express a group of genes, such as Gadd45alpha, whose overexpression can induce apoptosis, in response to hypoxia. Atf-2(-/-) MEFs also had a higher saturation density than wild-type cells and expressed lower levels of Maspin, the breast cancer tumor suppressor, which is also known to enhance cellular sensitivity to apoptotic stimuli. Atf-2(-/-) MEFs underwent a lower degree of apoptosis at high cell density than wild-type cells. Atf-2(+/-) mice were highly prone to mammary tumors that expressed reduced levels of Gadd45alpha and Maspin. The ATF-2 mRNA levels in human breast cancers were lower than those in normal breast tissue. Thus, ATF-2 acts as a tumor susceptibility gene of mammary tumors, at least partly, by activating a group of target genes, including Maspin and Gadd45alpha.

Topics: Activating Transcription Factor 2; Animals; Anisomycin; Apoptosis; Breast Neoplasms; Cell Cycle Proteins; Cell Hypoxia; Female; Fibroblasts; Genes, Reporter; Genes, Tumor Suppressor; Genetic Predisposition to Disease; Humans; Mammary Neoplasms, Animal; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Inbred CBA; Mice, Knockout; Nuclear Proteins; Nucleic Acid Synthesis Inhibitors; Oligonucleotide Array Sequence Analysis; RNA, Messenger; Serpins; Tumor Cells, Cultured

IRS-1 (Insulin Receptor Substrate-1) is an adaptor protein important for insulin and IGF-I receptor (Insulin-like Growth Factor-IR) transduction to downstream targets. One mechanism recently identified to downregulate IGF-I or insulin receptor signaling in diabetic models is IRS-1 Ser(312) phosphorylation. To date, the importance of this residue in cancer is unknown. This paper identifies mechanisms leading to Ser(312) regulation in MCF-7 breast cancer cells. Whereas IGF-I phosphorylation of IRS(312) is PI (phosphatidylinositol) 3-kinase dependent, anisomycin stress treatment requires JNK activation to induce phosphorylation of IRS(312). We show that both IGF-I and anisomycin stress treatment converge downstream onto mTOR (Mammalian Target of Rapamycin) and PKCdelta (Protein Kinase C-delta) to induce IRS-1 Ser(312) phosphorylation. mTOR associates with IRS-1 and is primarily required for Ser(312) phosphorylation in response to stress or IGF-I treatment. PKCdelta binds to mTOR and its activity is also important for stress or IGF-I mediated Ser(312) phosphorylation. Thus, mTOR and PKCdelta convey diverse signals to regulate IRS-1 function.

Topics: Anisomycin; Breast Neoplasms; Enzyme Activation; Female; Humans; Insulin Receptor Substrate Proteins; Insulin-Like Growth Factor I; JNK Mitogen-Activated Protein Kinases; MAP Kinase Kinase 4; Mitogen-Activated Protein Kinase Kinases; Oligonucleotides, Antisense; Phosphoproteins; Phosphorylation; Protein Kinase C; Protein Kinase C-delta; Protein Kinases; Protein Synthesis Inhibitors; Ribosomal Protein S6 Kinases, 70-kDa; Serine; Signal Transduction; TOR Serine-Threonine Kinases; Tumor Cells, Cultured; Tyrosine

Resistance to the antiestrogen tamoxifen is the main stumbling block for the success of breast cancer therapy. We focused our study on cellular alterations induced by a prolonged treatment with the active tamoxifen metabolite hydroxytamoxifen (OHT). We show that a prolonged OHT treatment (for up to 7 days) led to a progressive increase in the level of phosphorylated p44/42 mitogen activated kinase (MAP kinase) induced by 10(-7) M TPA stimulation, without any significant change in the protein level. This effect was also observed in MCF-7 cells grown first in medium containing dextran-coated charcoal-treated FCS (DCC medium) for 20 days prior to OHT treatment, indicating a specific effect of the antiestrogen and not an effect of estrogen deprivation. It was prevented by cotreatment with estradiol and not observed in the estrogen receptor negative HeLa cell line, suggesting that it was mediated by the estrogen receptor. TPA induced phosphorylation of MEK1/2 was also raised by OHT treatment, without any change in their protein level or Raf-1 and H-Ras levels. When the MCF-7R OHT resistant cell line was grown in antiestrogen containing medium, the level of phosphorylated p44/42 MAP kinase was also high but reversed when the antiestrogen was removed. The 2 other MAP kinase, JNK and P38 pathways were not affected in the same way by OHT treatment. In conclusion, our data reveal that a prolonged OHT treatment, by increasing p44/42 MAPK activity, affects a key step in the growth control of MCF-7 cells, although not sufficiently to overcome the growth inhibitory effect of the drug.

Topics: Anisomycin; Anti-Bacterial Agents; Breast Neoplasms; Estradiol; Estrogen Antagonists; Female; Fulvestrant; Genes, ras; Humans; JNK Mitogen-Activated Protein Kinases; MAP Kinase Kinase 4; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinase Kinases; Mitogen-Activated Protein Kinases; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Proto-Oncogene Proteins c-raf; Receptors, Estrogen; Recombinant Proteins; Tamoxifen; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

We examined transforming growth factor (TGF) alpha, epidermal growth factor (EGF) and EGF receptor (EGFR) expression and signaling in three drug resistant MCF-7 human breast cancer sublines and asked whether these pathways contribute to the drug resistance phenotype. In the resistant sublines, upregulation of both TGFalpha and EGFR mRNA was observed. In an apparent contrast with upregulated growth factor and receptor gene expression, the drug resistant sublines displayed a reduced growth rate. Defects in the EGFR signaling pathway cascade were found in all examined drug resistant sublines, including altered EGF-induced Shc, Raf-1, or mitogen-activated protein kinase phosphorylation. Induction of c-fos mRNA expression by EGF was impaired in the sublines compared to parental MCF-7 cells. In contrast, the induction of the stress-activated protein kinase activity was similar in both parental and drug resistant cells. Evaluating the link between the reduced growth rate and drug resistance, serum starvation experiments were performed. These studies demonstrated that a reduced proliferative activity resulted in a marked reduction in sensitivity to cytotoxic agents in the parental MCF-7 cells. We propose that the altered EGFR levels frequently observed in drug resistant breast cancer cells are associated with perturbations in the signaling pathway that mediate a reduced proliferative rate and thereby contribute to drug resistance.

Topics: Anisomycin; Antineoplastic Agents; Breast Neoplasms; Cell Division; Cell Line; DNA-Binding Proteins; Doxorubicin; Drug Resistance; Epidermal Growth Factor; ErbB Receptors; Humans; Immunoblotting; Mitogen-Activated Protein Kinase 9; Mitogen-Activated Protein Kinases; Paclitaxel; Phenotype; Precipitin Tests; Proto-Oncogene Proteins c-fos; RNA, Messenger; Signal Transduction; Transforming Growth Factor alpha; Tumor Cells, Cultured

In the human breast cancer cell line MCF-7, the nucleotides ATP gamma S and UTP, acting extracellularly through the purinergic receptor P2Y(2), lead to elevated intracellular calcium levels and increased proliferation. ATP gamma S and UTP treatment of MCF-7 cells activated transcription of the immediate early gene c-fos, an important component in the response to proliferative stimulation. c-fos induction was enhanced by co-treatment with ATP gamma S and a variety of proliferative agents including growth factors, tumour promoters and stress. Stimulation with ATP gamma S or epidermal growth factor (EGF) led to extracellular signal-regulated kinase (ERK) activation and phosphorylation of the transcription factors CREB and Elk-1. Co-stimulation synergistically activated fos expression and notably led to increased levels of ERK, CREB and EGF receptor phosphorylation, as well as hyperphosphorylation of ternary complex factor. Nevertheless, the ERK pathway does not fully account for this synergy, since fos induction was differentially sensitive to the MEK inhibitor U0126, indicating that these two agonists signal differently to this immediate early gene. Thus, extracellular nucleotides co-operate with growth factors to activate genes linked to the proliferative response in MCF-7 cells through activation of specific purinergic receptors, which thereby represent important potential targets for arresting the neoplastic progression of breast cancer cells.

Topics: Adenosine Triphosphate; Anisomycin; Breast Neoplasms; Carcinogens; Epidermal Growth Factor; Female; Gene Expression Regulation, Neoplastic; Genes, fos; Growth Substances; Humans; Parathyroid Hormone-Related Protein; Proteins; Signal Transduction; Tetradecanoylphorbol Acetate; Transcription, Genetic; Transforming Growth Factor beta; Tumor Cells, Cultured