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01.12.2017 | Research article | Ausgabe 1/2017 Open Access

Breast Cancer Research 1/2017

DCYTB is a predictor of outcome in breast cancer that functions via iron-independent mechanisms

Zeitschrift:
Breast Cancer Research > Ausgabe 1/2017
Autoren:
David J. Lemler, Miranda L. Lynch, Lia Tesfay, Zhiyong Deng, Bibbin T. Paul, Xiaohong Wang, Poornima Hegde, David H. Manz, Suzy V. Torti, Frank M. Torti
Wichtige Hinweise

Electronic supplementary material

The online version of this article (doi:10.​1186/​s13058-017-0814-9) contains supplementary material, which is available to authorized users.

Abstract

Background

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.

Methods

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.

Results

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.

Conclusions

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.
Zusatzmaterial
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)
13058_2017_814_MOESM1_ESM.pptx
Additional file 2: Supplemental Table. Table S1. Perturbed pathways identified by Signaling Pathway Impact Analysis (SPIA). (DOCX 15 kb)
13058_2017_814_MOESM2_ESM.docx
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