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. 2012 Feb;132(1):175-87.
doi: 10.1007/s10549-011-1536-9. Epub 2011 May 20.

Transcriptomic and proteomic profiling of KEAP1 disrupted and sulforaphane-treated human breast epithelial cells reveals common expression profiles

Abena S Agyeman  1 , Raghothama Chaerkady, Patrick G Shaw, Nancy E Davidson, Kala Visvanathan, Akhilesh Pandey, Thomas W Kensler
Affiliations

Transcriptomic and proteomic profiling of KEAP1 disrupted and sulforaphane-treated human breast epithelial cells reveals common expression profiles

Abena S Agyeman et al. Breast Cancer Res Treat. 2012 Feb.

Abstract

Sulforaphane (SFN), an isothiocyanate found in cruciferous vegetables, is a potent inhibitor of experimental mammary carcinogenesis and may be an effective, safe chemopreventive agent for use in humans. SFN acts in part on the Keap1/Nrf2 pathway to regulate a battery of cytoprotective genes. In this study, transcriptomic and proteomic changes in the estrogen receptor negative, non-tumorigenic human breast epithelial MCF10A cell line were analyzed following SFN treatment or KEAP1 knockdown with siRNA using microarray and stable isotopic labeling with amino acids in culture (SILAC), respectively. Changes in selected transcripts and proteins were confirmed by PCR and Western blot in MCF10A and MCF12A cells. There was strong correlation between the transcriptomic and proteomic responses in both the SFN treatment (R = 0.679, P < 0.05) and KEAP1 knockdown (R = 0.853, P < 0.05) experiments. Common pathways for SFN treatment and KEAP1 knockdown were xenobiotic metabolism and antioxidants, glutathione metabolism, carbohydrate metabolism, and NADH/NADPH regeneration. Moreover, these pathways were most prominent in both the transcriptomic and the proteomic analyses. The aldo-keto reductase family members, AKR1B10, AKR1C1, AKR1C2 and AKR1C3, as well as NQO1 and ALDH3A1, were highly upregulated at both the transcriptomic and the proteomic levels. Collectively, these studies served to identify potential biomarkers that can be used in clinical trials to investigate the initial pharmacodynamic action of SFN in the breast.

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Conflict of interest statement

Conflicts of interest: No potential conflicts of interest were disclosed.

Figures

Figure 1
Figure 1
Workflow for microarray and SILAC experiments. The vehicle used for SFN was acetonitrile. NTC=Non Targeting Control. LC-MS/MS = Liquid Chromatography tandem mass spectrometry. QTOF = Quadripole Time of Flight
Figure 2
Figure 2
Detailed workflow for SILAC experiments. The vehicle used for SFN was acetonitrile. Lys=Lysine. Arg=Arginine. Prototypical MS traces from LC-MS/MS are shown indicating the 6 Dalton (Da) shift between light and heavy labeled amino acid isotopes.
Figure 3
Figure 3
Right: Distribution of fold changes in proteins determined by SILAC between vehicle and SFN treated MCF-10A cells. Highlighted are the 96 upregulated and 26 downregulated proteins above and below a 1.5 fold change cut-off respectively. VC= Vehicle Control Left: Distribution of fold changes in proteins determined by SILAC between Non targeting Control and KEAP1 knockdown siRNA treated MCF-10A cells. Highlighted are the 50 upregulated and 76 downregulated proteins above and below a 1.5 fold change cut-off respectively. NTC=Non Targeting Control. KEAP1 KD= KEAP1 knockdown.
Figure 4
Figure 4
Western blots for proteins of interest from microarray and SILAC studies showing elevation of protein levels with SFN treatment and KEAP1 knockdown.
Figure 5
Figure 5
MS spectra from SILAC analyses of candidate biomarker proteins: (a) ALDH3A1:SFN Treatment, (b) ALDH3A1:KEAP1 Knockdown, (c) AKR1C1/2: SFN Treatment, (d) AKR1C1/2: KEAP1 Knockdown, (e) AKR1C3: SFN Treatment, (f) AKR1C3: KEAP1 knockdown, (g) NQO1: SFN Treatment and (h) NQO1: KEAP1 Knockdown
Figure 5
Figure 5
MS spectra from SILAC analyses of candidate biomarker proteins: (a) ALDH3A1:SFN Treatment, (b) ALDH3A1:KEAP1 Knockdown, (c) AKR1C1/2: SFN Treatment, (d) AKR1C1/2: KEAP1 Knockdown, (e) AKR1C3: SFN Treatment, (f) AKR1C3: KEAP1 knockdown, (g) NQO1: SFN Treatment and (h) NQO1: KEAP1 Knockdown
Figure 5
Figure 5
MS spectra from SILAC analyses of candidate biomarker proteins: (a) ALDH3A1:SFN Treatment, (b) ALDH3A1:KEAP1 Knockdown, (c) AKR1C1/2: SFN Treatment, (d) AKR1C1/2: KEAP1 Knockdown, (e) AKR1C3: SFN Treatment, (f) AKR1C3: KEAP1 knockdown, (g) NQO1: SFN Treatment and (h) NQO1: KEAP1 Knockdown
Figure 5
Figure 5
MS spectra from SILAC analyses of candidate biomarker proteins: (a) ALDH3A1:SFN Treatment, (b) ALDH3A1:KEAP1 Knockdown, (c) AKR1C1/2: SFN Treatment, (d) AKR1C1/2: KEAP1 Knockdown, (e) AKR1C3: SFN Treatment, (f) AKR1C3: KEAP1 knockdown, (g) NQO1: SFN Treatment and (h) NQO1: KEAP1 Knockdown
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