Discovery of IL-18 as a novel secreted protein contributing to doxorubicin resistance by comparative secretome analysis of MCF-7 and MCF-7/Dox

Abstract

Background: Resistance to chemotherapy is the major cause of failure in breast cancer treatment. Recent studies suggest that secreted proteins may play important roles in chemoresistance. We sought to systematically characterize secreted proteins associated with drug resistance, which may represent potential serum biomarkers or novel drug targets.

Methodology/principal findings: In the present work, we adopted the proteomic strategy of one-dimensional gel electrophoresis followed by liquid chromatography-tandem mass spectrometry to compare the secretome of MCF-7 and doxorubicin-resistant MCF-7/Dox. A total of 2,084 proteins were identified with at least two unique peptides in the conditioned media of two cell lines. By quantification with label-free spectral counting, 89 differentially expressed secreted proteins (DESPs) between the two cell lines were found. Among them, 57 DESPs were first found to be related to doxorubicin resistance in this work, including 24 extracellular matrix related proteins, 2 cytokines and 31 unclassified proteins. We focused on 13 novel DESPs with confirmed roles in tumor metastasis. Among them, the elevated expression of IL-18 in doxorubicin-resistant cell lines and breast tumor tissues was validated and its role in doxorubicin resistance was further confirmed by cell viability experiments in the presence or absence of this protein.

Conclusions/significance: Comparative analysis of the secretome of MCF-7 and MCF-7/Dox identified novel secreted proteins related to chemotherapy resistance. IL-18 was further validated to contribute to doxorubicin resistance, in addition to its confirmed role in breast cancer metastasis. Due to its dual roles in both drug resistance and tumor metastasis, IL-18 may represent a useful drug target for breast cancer therapy.

Figure 1. Workflow for the comparative secretome analysis of MCF-7 and MCF-7/Dox.

Illustration of the label-free approach to identify the differential proteins in the CM of MCF-7 and MCF-7/Dox cell lines.

Figure 2. Overview of proteins identified in the CM of MCF-7 and MCF-7/Dox and their subcellular localization.

Overlap of total proteins (A) and secreted proteins (B) identified in the CM of MCF-7 and MCF-7/Dox. (C) Subcellular localization of total proteins identified in the CM of MCF-7 and MCF-7/Dox. (D, E) Subcellular localization of proteins identified in the CM of MCF-7 and MCF-7/Dox, respectively.

Figure 3. Functional analysis and classification of DESPs.

(A) The functional analysis of DESPs between MCF-7 and MCF-7/Dox. (B) The classification of DESPs between MCF-7 and MCF-7/Dox.

Figure 4. Canonical pathways analysis of DESPs with potential dual functions in drug resistance and tumor metastasis.

32 DESPs with dual functions were analyzed by software IPA. The top 10 canonical pathways were mapped onto the DESPs, such as IGF-1 signaling, acute phase response signaling and IL-6 signaling etc. IL-6 and IL-18 were located as centers to link multiple signaling pathways.

Figure 5. Validation of the expression level of IL-18 and its role in drug resistance.

(A) The mRNA level of IL-18 in MCF-7, MCF-7/Dox and MCF-7/DoxH. The amount of IL-18 mRNA was normalized against the expression of GAPDH and the data were presented as mean ± SD (n = 3). **, p<0.01. (B) The concentration of IL-18 in the CM of MCF-7, MCF-7/Dox and MCF-7/DoxH. The data were presented as mean ± SD (n = 3). **, p<0.01. (C) The expression level of IL-18 in the lysates of 18 breast tumor tissues, including 12 drug-sensitive tissues and 6 drug-resistant tissues. The data were presented as mean ± SD (n = 3). **, p<0.01. (D) The doxorubicin dose dependent survival rate of MCF-7 in the absence or presence of 40 ng/mL rIL-18. Data were presented as mean ± SD (n = 3). **, p<0.01. (E) The effect of anti-IL-18 neutralization on the survival rate of MCF-7/Dox. Data were presented as mean ± SD (n = 3). *, p<0.05, **, p<0.01.