The online version of this article (doi:10.1186/s13058-017-0814-9) contains supplementary material, which is available to authorized users.
Duodenal cytochrome b (DCYTB) is a ferrireductase that functions together with divalent metal transporter 1 (DMT1) to mediate dietary iron reduction and uptake in the duodenum. DCYTB is also a member of a 16-gene iron regulatory gene signature (IRGS) that predicts metastasis-free survival in breast cancer patients. To better understand the relationship between DCYTB and breast cancer, we explored in detail the prognostic significance and molecular function of DCYTB in breast cancer.
The prognostic significance of DCYTB expression was evaluated using publicly available microarray data. Signaling Pathway Impact Analysis (SPIA) of microarray data was used to identify potential novel functions of DCYTB. The role of DCYTB was assessed using immunohistochemistry and measurements of iron uptake, iron metabolism, and FAK signaling.
High DCYTB expression was associated with prolonged survival in two large independent cohorts, together totaling 1610 patients (cohort #1, p = 1.6e-11, n = 741; cohort #2, p = 1.2e-05, n = 869; log-rank test) as well as in the Gene expression-based Outcome for Breast cancer Online (GOBO) cohort (p < 1.0e-05, n = 1379). High DCYTB expression was also associated with increased survival in homogeneously treated groups of patients who received either tamoxifen or chemotherapy. Immunohistochemistry revealed that DCYTB is localized on the plasma membrane of breast epithelial cells, and that expression is dramatically reduced in high-grade tumors. Surprisingly, neither overexpression nor knockdown of DCYTB affected levels of ferritin H, transferrin receptor, labile iron or total cellular iron in breast cancer cells. Because SPIA pathway analysis of patient microarray data revealed an association between DCYTB and the focal adhesion pathway, we examined the influence of DCYTB on FAK activation in breast cancer cells. These experiments reveal that DCYTB reduces adhesion and activation of focal adhesion kinase (FAK) and its adapter protein paxillin.
DCYTB is an important predictor of outcome and is associated with response to therapy in breast cancer patients. DCYTB does not affect intracellular iron in breast cancer cells. Instead, DCYTB may retard cancer progression by reducing activation of FAK, a kinase that plays a central role in tumor cell adhesion and metastasis.
Additional file 1: Supplemental Figures. Figure S1. DCYTB expression is higher in ER+ than ER- patients. Figure S2. DCYTB expression decreases with increased tumor grade. Figure S3. High DCYTB expression is associated with increased distant metastasis-free survival and reduced hazard in the GOBO combined breast tumor dataset. Figure S4. DCYTB predicts outcome independent of ER and LN status. Figure S5. Survival by molecular subtype in cohort #1. Figure S6. Increased DCYTB expression in molecular subtypes with better outcome in cohort #2. Figure S7. DCYTB expression is decreased in breast tumors. Figure S8. DCYTB protein is decreased in malignant breast tissue. Figure S9. DCYTB is expressed at higher levels in T47D cells than MCF7 cells. Figure S10. Induction and activity of Tet-on DCYTB expression vector. Figure S11. Modulation of DCYTB expression does not affect proliferation of cancerous breast cells. Figure S12. Knockdown of DCYTB expression does not affect progression through the cell cycle. Figure S13. Immunofluorescence staining of DCYTB or empty vector expressing MCF7 cells. (PPTX 4201 kb)
Hong CC, Ambrosone CB, Ahn J, Choi JY, McCullough ML, Stevens VL, Rodriguez C, Thun MJ, Calle EE. Genetic variability in iron-related oxidative stress pathways (Nrf2, NQ01, NOS3, and HO-1), iron intake, and risk of postmenopausal breast cancer. Cancer Epidemiol Biomarkers Prev. 2007;16(9):1784–94. CrossRefPubMed
Habashy HO, Powe DG, Staka CM, Rakha EA, Ball G, Green AR, Aleskandarany M, Paish EC, Douglas Macmillan R, Nicholson RI, et al. Transferrin receptor (CD71) is a marker of poor prognosis in breast cancer and can predict response to tamoxifen. Breast Cancer Res Treat. 2010;119(2):283–93. CrossRefPubMed
Yang DC, Wang F, Elliott RL, Head JF. Expression of transferrin receptor and ferritin H-chain mRNA are associated with clinical and histopathological prognostic indicators in breast cancer. Anticancer Res. 2001;21(1B):541–9. PubMed
Srivastava M, Duong LT, Fleming PJ. Cytochrome b561 catalyzes transmembrane electron transfer. J Biol Chem. 1984;259(13):8072–5. PubMed
College of American Pathologists. Protocol for the Examination of Specimens From Patients With Invasive Carcinoma of the Breast. 2016 [ http://www.cap.org/ShowProperty?nodePath=/UCMCon/Contribution%20Folders/WebContent/pdf/cp-breast-invasive-16protocol-3300.pdf].
Tarca AL, Kathri P, Draghici S. SPIA: Signaling Pathway Impact Analysis (SPIA) using combined evidence of pathway over-representation and unusual signaling perturbations. In: R package version 2.24.0; 2013.
R Development Core Team. R: A language and environment for statistical computing. Vienna: R Foundation for Statistical Computing; 2015. URL https://www.R-project.org/.
Martin M, Brase JC, Ruiz A, Prat A, Kronenwett R, Calvo L, Petry C, Bernard PS, Ruiz-Borrego M, Weber KE, et al. Prognostic ability of EndoPredict compared to research-based versions of the PAM50 risk of recurrence (ROR) scores in node-positive, estrogen receptor-positive, and HER2-negative breast cancer. A GEICAM/9906 sub-study. Breast Cancer Res Treat. 2016;156(1):81–9. CrossRefPubMedPubMedCentral
Stossi F, Barnett DH, Frasor J, Komm B, Lyttle CR, Katzenellenbogen BS. Transcriptional profiling of estrogen-regulated gene expression via estrogen receptor (ER) alpha or ERbeta in human osteosarcoma cells: distinct and common target genes for these receptors. Endocrinology. 2004;145(7):3473–86. CrossRefPubMed
Nguyen DH, Zhou T, Shu J, Mao J. Quantifying chromogen intensity in immunohistochemistry via reciprocal intensity. http://dx.doi.org/10.1038/protex.2013.097.
National Center for Biotechnology Information. Gene Expression Omnibus [ http://www.ncbi.nlm.nih.gov/gds/].
Leek JT, Evan J, Parker HS, Fertig EJ, Jaffe AE, Storey JD: sva: Surrogate Variable Analysis. In ., R package version 3.14.0. edn.
Therneau T. A Package for Survival Analysis in S. Version 2.38; 2015.
Therneau TM, Grambsch PM. Modeling Survival Data: Extending the Cox Model. New York: Springer; 2000. CrossRef
- DCYTB is a predictor of outcome in breast cancer that functions via iron-independent mechanisms
David J. Lemler
Miranda L. Lynch
Bibbin T. Paul
David H. Manz
Suzy V. Torti
Frank M. Torti
- BioMed Central
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