kn-93 and Breast-Neoplasms

Carcinoembryonic antigen-related cell adhesion molecule-1 (CEACAM1), a transmembrane protein, expressed on normal breast epithelial cells is down-regulated in breast cancer. Phosphorylation of Thr-457 on the short cytoplasmic domain isoform (CEACAM1-SF) that is predominant in normal epithelial cells is required for lumen formation in a three-dimensional model that involves apoptosis of the central acinar cells. Calmodulin kinase IID (CaMKIID) was selected as a candidate for the kinase required for Thr-457 phosphorylation from a gene chip analysis comparing genes up-regulated in MCF7 cells expressing wild type CEACAM1-SF compared with the T457A-mutated gene (Chen, C. J., Kirshner, J., Sherman, M. A., Hu, W., Nguyen, T., and Shively, J. E. (2007) J. Biol. Chem. 282, 5749-5760). Up-regulation of CaMKIID during lumen formation was confirmed by analysis of mRNA and protein levels. CaMKIID was able to phosphorylate a synthetic peptide corresponding to the cytoplasmic domain of CEACAM1-SF and was covalently bound to biotinylated and T457C-modified peptide in the presence of a kinase trap previously described by Shokat and co-workers (Maly, D. J., Allen, J. A., and Shokat, K. M. (2004) J. Am. Chem. Soc. 126, 9160-9161). When cell lysates from wild type-transfected MCF7 cells undergoing lumen formation were incubated with the peptide and kinase trap, a cross-linked band corresponding to CaMKIID was observed. When these cells were treated with an RNAi that inhibits CaMKIID expression, lumen formation was blocked by over 90%. We conclude that CaMKIID specifically phosphorylates Thr-457 on CEACAM1-SF, which in turn regulates the process of lumen formation via apoptosis of the central acinar cells.

Topics: Adenocarcinoma; Amino Acid Sequence; Antigens, CD; Apoptosis; Benzylamines; Breast Neoplasms; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Cell Adhesion Molecules; Cell Culture Techniques; Epithelial Cells; Female; Humans; Mammary Glands, Human; MCF-7 Cells; Molecular Sequence Data; Mutagenesis; Oligonucleotide Array Sequence Analysis; Phosphorylation; Protein Kinase Inhibitors; RNA, Small Interfering; Sulfonamides

Neutrophil gelatinase-associated lipocalin (NGAL, a.k.a Lnc2) is a member of the lipocalin family and has diverse roles. NGAL can stabilize matrix metalloproteinase-9 from autodegradation. NGAL is considered as a siderocalin that is important in the transport of iron. NGAL expression has also been associated with certain neoplasias and is implicated in the metastasis of breast cancer. In a previous study, we examined whether ectopic NGAL expression would alter the sensitivity of breast epithelial, breast and colorectal cancer cells to the effects of the chemotherapeutic drug doxorubicin. While abundant NGAL expression was detected in all the cells infected with a retrovirus encoding NGAL, this expression did not alter the sensitivity of these cells to doxorubicin as compared with empty vector-transduced cells. We were also interested in determining the effects of ectopic NGAL expression on the sensitivity to small-molecule inhibitors targeting key signaling molecules. Ectopic NGAL expression increased the sensitivity of MCF-7 breast cancer cells to EGFR, Bcl-2 and calmodulin kinase inhibitors as well as the natural plant product berberine. Furthermore, when suboptimal concentrations of certain inhibitors were combined with doxorubicin, a reduction in the doxorubicin IC 50 was frequently observed. An exception was observed when doxorubicin was combined with rapamycin, as doxorubicin suppressed the sensitivity of the NGAL-transduced MCF-7 cells to rapamycin when compared with the empty vector controls. In contrast, changes in the sensitivities of the NGAL-transduced HT-29 colorectal cancer cell line and the breast epithelial MCF-10A cell line were not detected compared with empty vector-transduced cells. Doxorubicin-resistant MCF-7/Dox (R) cells were examined in these experiments as a control drug-resistant line; it displayed increased sensitivity to EGFR and Bcl-2 inhibitors compared with empty vector transduced MCF-7 cells. These results indicate that NGAL expression can alter the sensitivity of certain cancer cells to small-molecule inhibitors, suggesting that patients whose tumors exhibit elevated NGAL expression or have become drug-resistant may display altered responses to certain small-molecule inhibitors.

Topics: Acute-Phase Proteins; Antibiotics, Antineoplastic; Benzylamines; Berberine; Biphenyl Compounds; Breast Neoplasms; Calcium-Calmodulin-Dependent Protein Kinases; Cell Line, Tumor; Doxorubicin; Drug Resistance, Neoplasm; ErbB Receptors; Female; Gene Expression; HT29 Cells; Humans; Lipocalin-2; Lipocalins; MCF-7 Cells; Nitrophenols; Piperazines; Protein Kinase Inhibitors; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Quinazolines; Sirolimus; Sulfonamides; Tyrphostins

Regulation of genes targeted by the ligand-activated aryl hydrocarbon receptor (AhR) has been shown to be controlled by calcium (Ca(2+)) changes induced by AhR agonists such as the environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The present study was designed to characterize this link between Ca(2+) and the AhR pathway. We report that fast elevation of intracellular Ca(2+) in TCDD-exposed mammary MCF-7 cells was associated with transient enhanced activity of the Ca(2+)/calmodulin (CaM)-dependent protein kinase (CaMK) pathway. Chemical inhibition of this pathway using the CaM antagonist W7 or the CaMK inhibitor KN-93 strongly reduced TCDD-mediated induction of the AhR target gene CYP1A1. Small interfering RNA (siRNA)-mediated knockdown expression of CaMKIalpha, one of the CaMK isoforms, similarly prevented CYP1A1 up-regulation. Both KN-93 and siRNA targeting CaMKIalpha were found to abolish TCDD-mediated activation of CYP1A1 promoter and TCDD-triggered nuclear import of AhR, a crucial step of the AhR signaling pathway. TCDD-mediated inductions of various AhR targets, such as the drug metabolizing CYP1B1, the cytokine interleukin-1beta, the chemokines interleukin-8 and CCL1, the adhesion molecule beta7 integrin, and the AhR repressor, were also prevented by KN-93 in human macrophages. Taken together, these data identified the Ca(2+)/CaM/CaMKIalpha pathway as an important contributing factor to AhR-mediated genomic response.

Topics: Benzylamines; Breast Neoplasms; Calcium; Calcium-Calmodulin-Dependent Protein Kinase Type 1; Calmodulin; Cell Line, Tumor; Cytochrome P-450 CYP1A1; Environmental Pollutants; Enzyme Inhibitors; Female; Genes, Reporter; Humans; Luciferases, Renilla; Polychlorinated Dibenzodioxins; Receptors, Aryl Hydrocarbon; RNA, Messenger; RNA, Small Interfering; Statistics as Topic; Sulfonamides; Up-Regulation

Calcium is universally required for cell growth and proliferation. Calmodulin is the main intracellular receptor for calcium. Although calcium and calmodulin are well known to be required for cell cycle regulation, the target pathways for their action remain poorly defined. Potential targets include the calcium/calmodulin-dependent kinases (CaM-K). The aim of this study was to determine the role of the CaM-Ks on cell proliferation and progress through the cell cycle in breast cancer cells. CaM-KI inhibition with either KN-93 or specific interfering RNA (siRNA) caused an arrest in the cell cycle in the human breast cancer cell line, MCF-7. This arrest occurred in the G(1) phase of the cell cycle. Supporting this finding, CaM-K inhibition using KN-93 also resulted in a reduction of cyclin D1 protein and pRb phosphorylation when cells were compared with control cultures. Furthermore, inhibition of the upstream activator of CaM-KI, CaM-KK, using siRNA also resulted in cell cycle arrest. In summary, CaM-KK and CaM-KI participate in the control of the G(0)-G(1) restriction check point of the cell cycle in human breast cancer cells. This arrest seems due to an inhibition in cyclin D1 synthesis and a reduction in pRb phosphorylation. To the best of our knowledge, this is the first time that CaM-KK has been reported to be involved in mammalian cell cycle regulation and that CaM-Ks are regulating breast cancer cell cycle.

Topics: Benzylamines; Breast Neoplasms; Calcium-Calmodulin-Dependent Protein Kinase Kinase; Calcium-Calmodulin-Dependent Protein Kinases; Cell Cycle; Cell Line, Tumor; Down-Regulation; Epithelial Cells; Humans; Protein Kinase Inhibitors; Protein Serine-Threonine Kinases; Sulfonamides