dorsomorphin and Breast-Neoplasms

Advanced stages of breast cancer are frequently complicated by bone metastases which cause substantial cancer-related morbidity and mortality. The Wnt-signaling antagonist Dickkopf-1 (DKK-1) has emerged as a crucial factor in the development and progression of osteolytic bone metastases. Although several signaling pathways have been implicated in promoting DKK-1 production in breast cancer cells, pharmacological interventions that interfere with tumor DKK-1 synthesis still remain scarce. In the current study, using an unbiased approach, we identified the small molecule Dorsomorphin as a potent suppressor of DKK-1 in several breast cancer cell lines (MDA-MB-231, MDA-Bone, MDA-MET and MCF7, respectively). Here, Dorsomorphin suppressed DKK-1 mRNA and protein production by 70 and 90%, respectively (p <0.001). Whereas bone morphogenic protein (BMP)- and AMP activated protein kinase (AMPK)-signaling are two well-established targets of Dorsomorphin, we show that neither pathway is essentially involved in facilitating its inhibitory effects on DKK-1. In summary, we identified Dorsomorphin as a potent pharmacological inhibitor of DKK-1 production in breast cancer cells. Whether Dorsomorphin reflects a valuable therapeutic agent in breast cancer warrants further investigations.

Topics: AMP-Activated Protein Kinases; Breast Neoplasms; Cell Line, Tumor; Female; Gene Expression Regulation, Neoplastic; Humans; Intercellular Signaling Peptides and Proteins; MCF-7 Cells; Protein Kinase Inhibitors; Pyrazoles; Pyrimidines; RNA, Messenger; Signal Transduction; Wnt Signaling Pathway

Previous studies have indicated that the sensitivity of breast cancer cells to tumor necrosis factor‑related apoptosis‑inducing ligand (TRAIL)‑induced apoptosis is associated with the expression of death receptors on the cell membrane. However, drug resistance limits the use of TRAIL in cancer therapy. Numerous studies have indicated that death receptors, which induce apoptosis, are upregulated by the endoplasmic reticulum (ER) stress response. 3‑Bromopyruvate (3‑BP), an anticancer agent, inhibits cell growth and induces apoptosis through interfering with glycolysis. In the present study, it was demonstrated that 3‑BP synergistically sensitized breast cancer cells to TRAIL‑induced apoptosis via the upregulation of death receptor 5 (DR5). Furthermore, we found that the protein levels of glucose‑related protein 78 (GRP78) and CCAAT‑enhancer‑binding protein homologous protein (CHOP) increased following treatment with 3‑BP. The expression of Bax (in MCF‑7 cells) and caspase‑3 (in MDA‑MB‑231 cells) increased following co‑treatment with 3‑BP and TRAIL, whereas the expression of the anti‑apoptotic protein Bcl‑2 decreased. In order to investigate the molecular mechanism regulating this effect, the expression of adenosine monophosphate‑activated protein kinase (AMPK), activated by 3‑BP, was determined. It was demonstrated that phosphorylated‑AMPK was upregulated following treatment with 3‑BP. Notably, Compound C, an AMPK inhibitor, reversed the effects of 3‑BP. Finally, a synergistic antitumor effect of 3‑BP and TRAIL was observed in MCF‑7 cell xenografts in nude mice. In conclusion, these results indicated that 3‑BP sensitized breast cancer cells to TRAIL via the AMPK‑mediated upregulation of DR5.

Topics: AMP-Activated Protein Kinases; Animals; Antineoplastic Agents; Apoptosis; Breast Neoplasms; Cell Proliferation; Drug Resistance, Neoplasm; Drug Synergism; Endoplasmic Reticulum Chaperone BiP; Endoplasmic Reticulum Stress; Female; Humans; MCF-7 Cells; Mice; Mice, Inbred BALB C; Mice, Nude; Phosphorylation; Pyrazoles; Pyrimidines; Pyruvates; Receptors, TNF-Related Apoptosis-Inducing Ligand; Recombinant Proteins; TNF-Related Apoptosis-Inducing Ligand; Up-Regulation; Xenograft Model Antitumor Assays

The presence of tumor cells in the circulation is associated with a higher risk of metastasis in patients with breast cancer. Circulating breast tumor cells use tubulin-based structures known as microtentacles (McTNs) to re-attach to endothelial cells and arrest in distant organs. McTN formation is dependent on the opposing cytoskeletal forces of stable microtubules and the actin network. AMP-activated protein kinase (AMPK) is a cellular metabolic regulator that can alter actin and microtubule organization in epithelial cells. We report that AMPK can regulate the cytoskeleton of breast cancer cells in both attached and suspended conditions. We tested the effects of AMPK on microtubule stability and the actin-severing protein, cofilin. AMPK inhibition with compound c increased both microtubule stability and cofilin activation, which also resulted in higher McTN formation and re-attachment. Conversely, AMPK activation with A-769662 decreased microtubule stability and cofilin activation with concurrent decreases in McTN formation and cell re-attachment. This data shows for the first time that AMPK shifts the balance of cytoskeletal forces in suspended breast cancer cells, which affect their ability to form McTNs and re-attach. These results support a model where AMPK activators may be used therapeutically to reduce the metastatic efficiency of breast tumor cells.

Topics: AMP-Activated Protein Kinases; Biphenyl Compounds; Breast Neoplasms; Cell Line, Tumor; Cytoskeleton; Female; Humans; MCF-7 Cells; Microtubules; Neoplasm Metastasis; Pyrazoles; Pyrimidines; Pyrones; Thiophenes

Increasing evidence supports the theory that tumor growth, homeostasis, and recurrence are dependent on a small subset of cells with stem cell properties, redefined cancer initiating cells (CICs) or cancer stem cells. Bone morphogenetic proteins (BMPs) are involved in cell-fate specification during embryogenesis, in the maintenance of developmental potency in adult stem cells and may contribute to sustain CIC populations in breast carcinoma. Using the mouse A17 cell model previously related to mesenchymal cancer stem cells and displaying properties of CICs, we investigated the role of BMPs in the control of breast cancer cell plasticity. We showed that an autocrine activation of BMP signaling is crucial for the maintenance of mesenchymal stem cell phenotype and tumorigenic potential of A17 cells. Pharmacological inhibition of BMP signaling cascade by Dorsomorphin resulted in the acquisition of epithelial-like traits by A17 cells, including expression of Citokeratin-18 and E-cadherin, through downregulation of Snail and Slug transcriptional factors and Cyclooxygenase-2 (COX2) expression, and in the loss of their stem-features and self-renewal ability. This phenotypic switch compromised A17 cell motility, invasiveness and in vitro tumor growth. These results reveal that BMPs are key molecules at the crossroad between stemness and cancer.

Topics: Animals; Bone Morphogenetic Protein 4; Bone Morphogenetic Protein Receptors, Type I; Bone Morphogenetic Protein Receptors, Type II; Bone Morphogenetic Proteins; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Cell Survival; Cyclooxygenase 2; Epithelial-Mesenchymal Transition; Female; Gene Expression Regulation; Humans; Inhibitor of Differentiation Protein 1; Mice; Neoplastic Stem Cells; Phenotype; Pyrazoles; Pyrimidines; Signal Transduction

Aberrant expression of mitotic checkpoint genes compromises mitotic checkpoint, leads to chromosome instability and tumorigenesis. However, the cell signals that control mitotic checkpoint gene expression have not been reported so far. In the present study we show that, in human breast cancer cells, chemical inhibition of Bone morphogenetic proteins (BMPs), but not Transforming Growth Factor-β (TGF-β), abrogates the mitotic arrest induced by nocodazole. Protein expression analysis reveals that inhibition of BMP signaling dramatically down regulates protein levels of mitotic checkpoint components BUB3, Hec1, TTK and MAD2, but inhibition of TGF-β has relatively minor effect on the expression of these proteins. Activation of BMP signaling specifically up regulates BUB3, and activation of Activin A signaling globally down regulates these proteins level. Furthermore, overexpressing MAD2, TTK, BUB3 or Hec1 significantly rescues the mitotic arrest defect caused by BMP inhibition. Our results demonstrated for the first time that TGF-β family cytokines are cellular signals regulating mitotic checkpoint and perturbations in intrinsic BMP signaling could lead to suppression of mitotic checkpoint signaling by downregulating key checkpoint proteins. The results suggest a possible mechanism by which dysregulation of TGF-β signaling causes mitotic checkpoint defects and drives tumorigenesis. The finding also provides a potential and more specific strategy for cancer prevention by targeting BMP and mitotic checkpoint connection.

Topics: Activins; Benzamides; Bone Morphogenetic Proteins; Breast Neoplasms; Calcium-Binding Proteins; Cell Cycle Checkpoints; Cell Cycle Proteins; Cell Line, Tumor; Cell Transformation, Neoplastic; Cytoskeletal Proteins; Dioxoles; Female; Gene Expression Regulation, Neoplastic; HEK293 Cells; Humans; Mad2 Proteins; Nocodazole; Nuclear Proteins; Plasmids; Poly-ADP-Ribose Binding Proteins; Protein Kinase Inhibitors; Protein Serine-Threonine Kinases; Protein-Tyrosine Kinases; Pyrazoles; Pyrimidines; Repressor Proteins; Signal Transduction; Transfection; Transforming Growth Factor beta

Adenosine monophosphate (AMP)-activated kinase (AMPK) is a molecular energy sensor regulated by the tumor suppressor LKB1. Starvation and growth factors activate AMPK through the DNA damage sensor ataxia-telangiectasia mutated (ATM). We explored the regulation of AMPK by ionizing radiation (IR) and its role as a target for radiosensitization of human cancer cells.. Lung, prostate, and breast cancer cells were treated with IR (2-8 Gy) after incubation with either ATM or AMPK inhibitors or the AMPK activator metformin. Then, cells were subjected to either lysis and immunoblotting, immunofluorescence microscopy, clonogenic survival assays, or cell cycle analysis.. IR induced a robust phosphorylation and activation of AMPK in all tumor cells, independent of LKB1. IR activated AMPK first in the nucleus, and this extended later into cytoplasm. The ATM inhibitor KU-55933 blocked IR activation of AMPK. AMPK inhibition with Compound C or anti-AMPK alpha subunit small interfering RNA (siRNA) blocked IR induction of the cell cycle regulators p53 and p21(waf/cip) as well as the IR-induced G2/M arrest. Compound C caused resistance to IR, increasing the surviving fraction after 2 Gy, but the anti-diabetic drug metformin enhanced IR activation of AMPK and lowered the surviving fraction after 2 Gy further.. We provide evidence that IR activates AMPK in human cancer cells in an LKB1-independent manner, leading to induction of p21(waf/cip) and regulation of the cell cycle and survival. AMPK appears to (1) participate in an ATM-AMPK-p21(waf/cip) pathway, (2) be involved in regulation of the IR-induced G2/M checkpoint, and (3) may be targeted by metformin to enhance IR responses.

Topics: AMP-Activated Protein Kinases; Ataxia Telangiectasia Mutated Proteins; Breast Neoplasms; Cell Cycle Proteins; Cell Line, Tumor; Cell Survival; Cyclin-Dependent Kinase Inhibitor p21; DNA-Binding Proteins; Enzyme Activation; Female; G2 Phase; Humans; Lung Neoplasms; Male; Metformin; Morpholines; Phosphorylation; Prostatic Neoplasms; Protein Serine-Threonine Kinases; Pyrazoles; Pyrimidines; Pyrones; Radiation Tolerance; RNA, Small Interfering; Tumor Suppressor Protein p53; Tumor Suppressor Proteins

Compound C is commonly used as an inhibitor of AMP-activated protein kinase (AMPK), which serves as a key energy sensor in cells. In this study, we found that Compound C treatment of MCF7 cells led to Bax redistribution from the cytoplasm to mitochondria and cell death. However, this effect does not involve AMPK. In addition, we found that treatment with this compound leads to an enhanced ceramide production. Analyses by quantitative PCR and ceramide synthase activity assay suggest that ceramide synthase 5 (LASS/CerS 5) is involved in Compound C-induced ceramide upregulation. Downregulation of LASS/CerS 5 was found to attenuate Compound C-mediated ceramide production, Bax redistribution, and cell death.

Topics: AMP-Activated Protein Kinases; Apoptosis; bcl-2-Associated X Protein; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Ceramides; Humans; Mitochondria; Oxidoreductases; Polymerase Chain Reaction; Protein Transport; Pyrazoles; Pyrimidines; Sphingosine N-Acyltransferase