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

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 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 proteomic level. 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.

Oxidative Stress and Response to Thymidylate Synthase-Targeted Antimetabolites

“…NRF2 is a member of the cap 'n collar family of transcription factors that controls expression of genes conferring protection against oxidative and electrophilic stress (Baird and Dinkova-Kostova, 2011;Jaramillo and Zhang, 2013). Table 2 lists 27 transcripts from the microarray whose expression levels have been identified in the literature as regulated by NRF2 (Agyeman et al, 2012;Suzuki et al, 2013;Choi et al, 2014;Hayes and Dinkova-Kostova, 2014). We tested 15 of these by qRT-PCR and confirmed all as inducible by FdUrd (Table 2).…”

“…6B) Genes that did not show up in the microarray analysis but are known from the literature to be NRF2 targets (Agyeman et al, 2012;Suzuki et al, 2013;Choi et al, 2014;Hayes and Dinkova-Kostova, 2014) were tested for response to TYMS inhibitors by qRT-PCR. The fold-change represents the extent of induction by 10 mM FdUrd (with 10 mM folinic acid) for 24 hours before harvesting.…”

“…Under normal circumstances, NRF2 is maintained at low levels within the cell, a status maintained by the transcription factor's TABLE 2 Identification of FUra-inducible NRF2 target genes by gene expression profiling NRF2 target genes identified in the whole genome microarray analysis (Supplemental Table 2) are listed. Classification of each gene as an NRF2 target is based upon the literature (Agyeman et al, 2012;Suzuki et al, 2013;Choi et al, 2014;Hayes and Dinkova-Kostova, 2014). The fold change represents the extent of induction by exposure to 10 mM FUra for 24 hours (see text).…”

Keap1–Nrf2 signalling in pancreatic cancer

“…This regulator was originally identified by yeast two-hybrid analysis (Itoh et al, 1999), and was confirmed as a repressor by the finding that Nrf2 accumulated in the nucleus and Nrf2-target genes were upregulated in Keap1 gene knockout mice . Moreover, knockdown of Keap1 in human cell lines similarly increases expression of Nrf2-target genes (Devling et al, 2005;Agyeman et al, 2012). In the cytoplasm, Keap1 may be associated with the cytoskeleton and interacts with cullin-3 (via its BTB domain) and the Neh2 domain of Nrf2 (via its Kelch-repeat domain) (McMahon et al, 2003;Nioi and Hayes, 2004).…”

Alcohol Causes Alveolar Epithelial Oxidative Stress by Inhibiting the Nuclear Factor (Erythroid-Derived 2)–Like 2–Antioxidant Response Element Signaling Pathway

“…It is still not clear how SPN stimulates and/or activates the Nrf2-ARE pathway. It has been reported that SPN interacts with cysteines in Keap1, and consequently disrupts Keap1 ubiquitination and degradation of Nrf2 (22). Therefore, it appears that, at least within this context, SPN either has additional effects on Nrf2 expression or that its ability to stabilize Nrf2 promotes a feedback mechanism to induce the expression of more Nrf2.…”