Dataset Information

Characterization of the Interactions between Calmodulin and Death Receptor 5 in Triple-negative and Estrogen Receptor-positive Breast Cancer Cells: AN INTEGRATED EXPERIMENTAL AND COMPUTATIONAL STUDY.

ABSTRACT: Activation of death receptor-5 (DR5) leads to the formation of death inducing signaling complex (DISC) for apoptotic signaling. Targeting DR5 to induce breast cancer apoptosis is a promising strategy to circumvent drug resistance and present a target for breast cancer treatment. Calmodulin (CaM) has been shown to regulate DR5-mediated apoptotic signaling, however, its mechanism remains unknown. In this study, we characterized CaM and DR5 interactions in breast cancer cells with integrated experimental and computational approaches. Results show that CaM directly binds to DR5 in a calcium dependent manner in breast cancer cells. The direct interaction of CaM with DR5 is localized at DR5 death domain. We have predicted and verified the CaM-binding site in DR5 being (354)WEPLMRKLGL(363) that is located at the ?2 helix and the loop between ?2 helix and ?3 helix of DR5 DD. The residues of Trp-354, Arg-359, Glu-355, Leu-363, and Glu-367 in DR5 death domain that are important for DR5 recruitment of FADD and caspase-8 for DISC formation to signal apoptosis also play an important role for CaM-DR5 binding. The changed electrostatic potential distribution in the CaM-binding site in DR5 DD by the point mutations of W354A, E355K, R359A, L363N, or E367K in DR5 DD could directly contribute to the experimentally observed decreased CaM-DR5 binding by the point mutations of the key residues in DR5 DD. Results from this study provide a key step for the further investigation of the role of CaM-DR5 binding in DR5-mediated DISC formation for apoptosis in breast cancer cells.

SUBMITTER: Fancy RM

PROVIDER: S-EPMC4933462 | biostudies-literature | 2016 Jun

REPOSITORIES: biostudies-literature

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Characterization of the Interactions between Calmodulin and Death Receptor 5 in Triple-negative and Estrogen Receptor-positive Breast Cancer Cells: AN INTEGRATED EXPERIMENTAL AND COMPUTATIONAL STUDY.

Fancy Romone M RM Wang Lingyun L Zeng Qinghua Q Wang Hong H Zhou Tong T Buchsbaum Donald J DJ Song Yuhua Y

The Journal of biological chemistry 20160422 24

Activation of death receptor-5 (DR5) leads to the formation of death inducing signaling complex (DISC) for apoptotic signaling. Targeting DR5 to induce breast cancer apoptosis is a promising strategy to circumvent drug resistance and present a target for breast cancer treatment. Calmodulin (CaM) has been shown to regulate DR5-mediated apoptotic signaling, however, its mechanism remains unknown. In this study, we characterized CaM and DR5 interactions in breast cancer cells with integrated experi ...[more]

PMID: 27129269

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Project description:The Fas death receptor-activated death-inducing signaling complex (DISC) regulates apoptosis in many normal and cancer cells. Qualitative biochemical experiments demonstrate that calmodulin (CaM) binds to the death domain of Fas. The interaction between CaM and Fas regulates Fas-mediated DISC formation. A quantitative understanding of the interaction between CaM and Fas is important for the optimal design of antagonists for CaM or Fas to regulate the CaM-Fas interaction, thus modulating Fas-mediated DISC formation and apoptosis. The V254N mutation of the Fas death domain (Fas DD) is analogous to an identified mutant allele of Fas in lpr-cg mice that have a deficiency in Fas-mediated apoptosis. In this study, the interactions of CaM with the Fas DD wild type (Fas DD WT) and with the Fas DD V254N mutant were characterized using isothermal titration calorimetry (ITC), circular dichroism spectroscopy (CD), and molecular dynamics (MD) simulations. ITC results reveal an endothermic binding characteristic and an entropy-driven interaction of CaM with Fas DD WT or with Fas DD V254N. The Fas DD V254N mutation decreased the association constant (Ka) for CaM-Fas DD binding from (1.79 ± 0.20) × 10(6) to (0.88 ± 0.14) × 10(6) M(-1) and slightly increased a standard state Gibbs free energy (ΔG°) for CaM-Fas DD binding from -8.87 ± 0.07 to -8.43 ± 0.10 kcal/mol. CD secondary structure analysis and MD simulation results did not show significant secondary structural changes of the Fas DD caused by the V254N mutation. The conformational and dynamical motion analyses, the analyses of hydrogen bond formation within the CaM binding region, the contact numbers of each residue, and the electrostatic potential for the CaM binding region based on MD simulations demonstrated changes caused by the Fas DD V254N mutation. These changes caused by the Fas DD V254N mutation could affect the van der Waals interactions and electrostatic interactions between CaM and Fas DD, thereby affecting CaM-Fas DD interactions. Results from this study characterize CaM-Fas DD interactions in a quantitative way, providing structural and thermodynamic evidence of the role of the Fas DD V254N mutation in the CaM-Fas DD interaction. Furthermore, the results could help to identify novel strategies for regulating CaM-Fas DD interactions and Fas DD conformation and thus to modulate Fas-mediated DISC formation and thus Fas-mediated apoptosis.

Project description:Estrogen receptor-positive and human epidermal growth factor receptor 2-negative (ER+ HER2- ) breast cancer accounts for ~ 60-70% of all cases of invasive breast carcinoma. High-grade ER+ HER2- tumors respond poorly to endocrine therapy. In this study, we systematically analyzed clinical and multi-omics data to find potential strategies for personalized therapy of patients with high-grade ER+ HER2- disease. Six different cohorts were analyzed, for which multi-omics data were available. Grade III ER+ HER2- cases harbored higher proportions of large tumor size (> 5 cm), lymph node metastasis, chemotherapy use, and luminal B subtypes defined by PAM50, as compared with grade I/II tumors. DNA methylation (HM450) data and methylation-specific PCR indicated that the cg18629132 locus in the MKI67 promoter was hypermethylated in grade I/II cases and normal tissue, but hypomethylated in grade III cases or triple-negative breast cancer, resulting in higher expression of MKI67. Mutations in ESR1 and TP53 were detected in post-endocrine treatment metastatic samples at a higher rate than in treatment-naive tumors in grade III cases. We identified 42 and 20 focal copy number events in nonmetastatic and metastatic high-grade ER+ HER2- cases, respectively, with either MYC or MDM2 amplification representing an independent prognostic event in grade III cases. Transcriptional profiling within grade III tumors highlighted ER signaling downregulation and upregulation of immune-related pathways in non-luminal-like tumors defined by PAM50. Recursive partitioning analysis was employed to construct a decision tree of an endocrine-resistant subgroup (GATA3-negative and AGR-negative) of two genes that was validated by immunohistochemistry in a Chinese cohort. All together, these data suggest that grade III ER+ HER2- tumors have distinct clinical and molecular characteristics compared with low-grade tumors, particularly in cases with non-luminal-like biology. Due to the dismal prognosis in this group, clinical trials are warranted to test the efficacy of potential novel therapies.

Project description:PURPOSE:A subset of estrogen receptor-positive (ER-positive) breast cancer (BC) contains high levels of tumor-infiltrating lymphocytes (TILs), similar to triple-negative BC (TNBC). The majority of immuno-oncology trials target TNBCs because of the greater proportion of TIL-rich TNBCs. The extent to which the immune microenvironments of immune-rich ER-positive BC and TNBC differ is unknown. PATIENTS AND METHODS:RNA sequencing data from The Cancer Genome Atlas (TCGA; n = 697 ER-positive BCs; n = 191 TNBCs) were used for discovery; microarray expression data from Molecular Taxonomy of Breast Cancer International Consortium (METABRIC; n = 1,186 ER-positive BCs; n = 297 TNBCs) was used for validation. Patients in the top 25th percentile of a previously published total TIL metagene score distribution were considered immune rich. We compared expression of immune cell markers, immune function metagenes, and immuno-oncology therapeutic targets among immune-rich subtypes. RESULTS:Relative fractions of resting mast cells (TCGA P adj = .009; METABRIC P adj = 4.09E-15), CD8+ T cells (TCGA P adj = .015; METABRIC P adj = 0.390), and M2-like macrophages (TCGA P adj= 4.68E-05; METABRIC P adj = .435) were higher in immune-rich ER-positive BCs, but M0-like macrophages (TCGA P adj = 0.015; METABRIC P adj = .004) and M1-like macrophages (TCGA P adj = 9.39E-08; METABRIC P adj = 6.24E-11) were higher in immune-rich TNBCs. Ninety-one immune-related genes (eg, CXCL14, CSF3R, TGF-B3, LRRC32/GARP, TGFB-R2) and a transforming growth factor ? (TGF-?) response metagene were significantly overexpressed in immune-rich ER-positive BCs, whereas 41 immune-related genes (eg, IFNG, PD-L1, CTLA4, MAGEA4) were overexpressed in immune-rich TNBCs in both discovery and validation data sets. TGF-? pathway member genes correlated negatively with expression of immune activation markers (IFNG, granzyme-B, perforin) and positively with M2-like macrophages (IL4, IL10, and MMP9) and regulatory T-cell (FOXP3) markers in both subtypes. CONCLUSION:Different immunotherapy strategies may be optimal in immune-rich ER-positive BC and TNBC. Drugs targeting the TGF-? pathway and M2-like macrophages are promising strategies in immune-rich ER-positive BCs to augment antitumor immunity.

Dataset Information

Characterization of the Interactions between Calmodulin and Death Receptor 5 in Triple-negative and Estrogen Receptor-positive Breast Cancer Cells: AN INTEGRATED EXPERIMENTAL AND COMPUTATIONAL STUDY.

Publications

Characterization of the Interactions between Calmodulin and Death Receptor 5 in Triple-negative and Estrogen Receptor-positive Breast Cancer Cells: AN INTEGRATED EXPERIMENTAL AND COMPUTATIONAL STUDY.

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