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. 2019 Dec 6;11(23):11329-11346.
doi: 10.18632/aging.102531. Epub 2019 Dec 6.

Interplay of MKP-1 and Nrf2 drives tumor growth and drug resistance in non-small cell lung cancer

Hongyan Wang  1 , Kaihua Liu  1 , Zhexu Chi  2 , Xihang Zhou  2 , Guoping Ren  3 , Ren Zhou  4   5 , Yinyan Li  1 , Xiuwen Tang  2 , Xiu Jun Wang  1
Affiliations

Interplay of MKP-1 and Nrf2 drives tumor growth and drug resistance in non-small cell lung cancer

Hongyan Wang et al. Aging (Albany NY). .

Abstract

Alterations in KEAP1/ NF-E2 p45-related factor 2 (NFE2L2/Nrf2) signaling pathway have been reported in 23% lung adenocarcinoma patients, suggesting that deregulation of the pathway is a major cancer driver. Here we report that mitogen-activated protein (MAP) kinase phosphatase 1 (MKP-1) drives tumor growth and drug resistance by up regulating transcription factor Nrf2. In non-small cell lung cancer (NSCLC) cells and xenografts, MKP-1 knockdown triggered the down-regulation of the metabolic enzymes and cytoprotective proteins, which are the target genes of Nrf2. Consequently, the cell growth was markedly inhibited with decrease of tumor metabolisms and GSH contents. Moreover, MKP-1 silencing sensitized NSCLC cells to cisplatin treatment. Mechanistically, MKP-1 inhibited the ubiquitylation of Nrf2 via a direct interaction with the transcription factor. Nrf2 was hence stabilized and its transcriptional program was activated. Notably, Nrf2 elevated MKP-1 expression at transcriptional level. In human lung adenoma tumor samples, high levels of expression of MKP-1, Nrf2, and its target gene heme oxygenase 1 were closely correlated. Thus, MKP-1 and Nrf2 form a forward feedback loop in lung cancer cells, which stabilizing and activating Nrf2 to promote anabolic metabolism and GSH biosynthesis. This study uncovers a novel role of MKP-1 in the malignant evolution of cancers.

Keywords: Nrf2; lung cancer; mitogen-activated protein kinase phosphatase 1.

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Conflict of interest statement

CONFLICTS OF INTEREST: The authors have declared no conflicts of interests.

Figures

Figure 1
Figure 1
MKP-1 regulates the proliferation and drug resistance of A549 NSCLC cells. Two stable MKP-1-knockdown cell lines, siMKP1-C1 and siMKP1-C2, were generated after stable transfection with the pGFP-V-RS-MKP-1 plasmid into A549 cells. siCon expressing empty pGFP-V-RS vector was used as a negative control. (A) MKP-1 mRNA levels in siCon, siMKP1-C1, and siMKP1-C2 cells as determined by Taqman RT-PCR. The 18S rRNA level was used as an internal control and the value for siCon cells was set at 100%. Values are mean ± SD, n = 3. (B) Immunoblots of whole-cell lysates probed with anti-MKP-1, anti-Nrf2 or anti-actin. The relative levels of MKP-1 and Nrf2 normalized to actin are shown above each lane. Blots are representative three separate experiments. (C) Knockdown of MKP-1 reduces cell migration. Scratch assay images of siMKP1-C1 (b and e), siMKP1-C2 (c and f) and siCon cells (a and d) acquired at 0 (a-c) and 24 h (d-f). Red lines define the areas lacking cells. Statistics are shown in (g). Values are mean ± SD, n = 3. (D) MKP-1 promotes motility of NSCLC cells. Images of transwell migration assays of siMKP1-C1 (b), siMKP1-C2 (c), and siCon cells (a). (d) Statistics for three experiments. The number of siCon cells was set at 100%. Values are mean ± SD, n = 3. (E) Knockdown of MKP-1 decreases cell proliferation. Cells were cultured for 24, 48, or 72 h and the numbers determined by MTS assays. The value for the same cells at 0 h was set at 1. Values are mean ± SD, n = 3. (F) Knockdown of MKP-1 increases sensitivity to cisplatin in NSCLC cells. siMKP1-C1, siMKP1-C2 and siCon cells were exposed to cisplatin (0–25 μg/ml) for 48 h. The cell viability was determined by MTT method. The value of DMSO treatment was set at 1. Values are means ± SD, n = 3. *p <0.05, **p <0.01.
Figure 2
Figure 2
MKP-1 regulates metabolism of A549 NSCLC cells and promotes tumor growth. (A) Representative images and weights of tumors 6 weeks after subcutaneous injection of siMKP1-C1, siMKP1-C2 or siCon cells into Nu/Nu mice. Values are mean ± SD, n = 8. (B) MKP-1 knockdown alters glucose and glutamine metabolism in A549 Cells. siMKP1-C1, siMKP1-C2 and siCon cells were cultured for three days before glucose consumption, lactate production, citrate, NADPH, G6PD activity, and GSH levels were determined. The value for culture medium at day 0 was set at 100% for the analysis of glucose consumption. The value for siCon cells was set at 100% for the analysis of GSH levels. Values are mean ± SD, n = 3, *p <0.05, **p <0.01. (C). MKP-1 regulates glucose and glutamine metabolism in NSCLC cells. Metabolite abbreviations: G6P, glucose 6-phosphate; F6P, fructose 6-phosphate; F1,6P, fructose 1,6-bis-phosphate; GA3P, glyceraldehyde 3-phosphate; 3-PG, 3-phosphoglycerate; PEP, phosphoenolpyruvate; 6-P-Gl, 6-phosphogluconolactone; 6-PG, 6-phosphogluconate; R-5-P, ribulose5-phosphate; PRPP, 5-phosphoribosyl-α-1-pyrophosphate; 5-PRA, b-5-phosphorybosylamine; GSH, reduced glutathione. The metabolic enzymes regulated by MKP-1/Nrf2 in glucose and glutamine metabolisms, are shown in red. G6PD, glucose-6-phosphate dehydrogenase; GCLC, glutamate-cysteine ligase, catalytic subunit; IDH1, isocitrate dehydrogenase 1; ME1, malic enzyme 1; MTHFD2, methylenetetrahydrofolate dehydrogenase 2; PGD, 6-phosphogluconate dehydrogenase; PPAT, phosphoribosyl pyrophosphate amidotransferase; TALDO1, transaldolase1; TKT, transketolase. The mitochondrion is shown in gray.
Figure 3
Figure 3
MKP-1 regulates Nrf2 and its target genes in NSCLC xenograft tumors. (A) and (B) Knockdown of MKP-1 reduces the expression of ARE-driven genes in NSCLC xenograft tumors. siMKP1-C1 or siCon cells were injected subcutaneously into Nu/Nu mice and the tumors were removed 6 weeks later. (A) mRNA levels of Nrf2 target genes determined by RT-PCR. The level of 18S rRNA was used as internal control. The value for siCon was set at 100%. Values are mean of three single tumours ± SD, n = 3 (B) Western immunoblots of the expression of MKP-1, Nrf2, and ARE-driven genes with antibodies against the indicated proteins. Each lane represents a single tumor. The relative levels of MKP-1, Nrf2, and ARE-driven genes normalized to actin are shown in right panel. The value for siCon was set at 1. Values are mean of three single tumours ± SD, n = 3. Blots are representative at least three separate experiments. (C) Knockdown of MKP-1 by siRNA against MKP-1 reduces the expression of Nrf2 protein and its target genes in A549 and H460 NSCLC cells. A549 and H460 cells were transfected with each of the MKP-1 siRNAs to Target 1, Target 2, or Target 3. Scrambled siRNA was transfected as negative control. Cells were harvested 48 h later and analyzed by Western immunoblotting with antibodies against the indicated proteins. The relative levels of MKP-1, Nrf2, and ARE-driven genes normalized to actin are shown above each lane. The value for scrambled siRNA-transfected cells was set at 1. Blots are representative at least three separate experiments. **p <0.01 (D) Overexpression MKP-1 increases the expression of Nrf2 protein and ARE-driven genes in A549 cells. (a) A549 cells were transfected with pEGFP-MKP1 or pEGFP vector 24 h before the cells were harvested. Nuclear extracts were probed by immunoblot with anti-MKP-1, anti-Nrf2, or anti-Lamin B1. The relative levels of MKP-1 or Nrf2 normalized to lamin B1 are shown above each lane. The value for pEGFP-transfected cells was set at 1. (b) Luciferase activity in A549 cells transfected with pEGFP, or pEGFP-MKP1. Co-transfections were performed with pGL-GSTA2.41bp-ARE reporter vector and pRL-TK. Dual luciferase activities were analysed. The value for cells transfected with pEGFP plus pGL-GSTA2.41bp-ARE reporter vector and pRL-TK was set at 1. Data are presented as the mean ± SD of triplicate experiments. (E) MKP-1 regulates Nrf2 ubiquitination. A549 cells were transfected with scrambled siRNA or MKP-1 siRNA for 48 h. The cells were exposed to MG132 (20 μM) for 4 h before whole-cell lysates were harvested and subjected to immunoprecipitation with Nrf2. After washing, the immunoprecipitates (Beads) were probed by immunoblotting with anti-UB. The input represents 10% of the total amount of cell lysate use for immunoprecipitation. Results are representative of three separate experiments. *p <0.05, **p < 0.01.
Figure 4
Figure 4
MKP-1 interacts with Nrf2. (A) Endogenous MKP-1 and Nrf2 interacted in A549 cells. Cell lysate from A549 cells was immunoprecipitated with antibody specific to MKP-1 or Nrf2. IgG was used as negative control. After washing, the immunoprecipitates were analysed by immunoblotting with antibody specific to Nrf2 or MKP-1. Input, 10% of the cell lysate used for immunoprecipitation. (B) Endogenous MKP-1 co-localised with Nrf2 in A549 and H460 cells. Cells were grown on cover-slips and fixed. Indirect immunofluorescence staining was performed to visualize endogenous MKP-1 using a primary rabbit antibody against MKP-1, and followed by Texas Red goat anti-rabbit secondary antibody. The endogenous Nrf2 was visualized using a primary mouse antibody against Nrf2 and followed by EGFP anti-mouse secondary antibody. The endogenous MKP-1 and Nrf2 are shown in red and green, respectively. Nuclei were stained with DAPI (blue). The scale bars represent 10 μm. Results are representative of three separate experiments.
Figure 5
Figure 5
MKP-1 is an Nrf2 target gene. (A) Knockdown of Nrf2 reduced the expression of MKP-1 in A549 cells. siNrf2-C27 cells derived from A549 cells stably expressed siRNA against Nrf2. (a) mRNA levels of MKP-1 in siNrf2-C27 and siGFP-C5 cells were determined by RT-PCR. The level of 18S rRNA was used as internal control. The value for siCon cells was set at 100%. Data are presented as the mean ± SD of triplicate experiments. (b) MKP-1 protein expression in siNrf2-C27 and siGFP-C5 cells was determined by Western immunoblotting with anti-MKP-1. The relative levels of MKP-1 normalized to actin are shown above each lane. The value for siGFP-C5 cells was set at 1. (B) Knockdown of Nrf2 decreased the mRNA and protein levels of MKP-1 in H460 cells. H460 cells were transiently transfected with siRNA against Nrf2. (a) Total RNAs were harvested 24 h later. The mRNA levels of Nrf2 and MKP-1 were measured by RT-PCR. The level of 18S rRNA was used as internal control. The value for scrambled siRNA was set at 100%. (b) MKP-1 protein expression in cells transfected with scrambled siRNA or Nrf2-siRNA cells was determined by Western immunoblotting with anti-MKP-1. The relative levels of MKP-1 normalized to actin are shown above each lane. The value for scrambled siRNA-transfected cells was set at 1. Blots in 5Ab and 5Bb are representative at least three separate experiments. Data in 5Aa and 5Ba are presented as the mean ± SD of triplicate experiments (*p <0.05, **p <0.01).
Figure 6
Figure 6
Association of the expression of Nrf2, MKP-1, and HO-1 in human lung adenoma tissues. Images of immunochemical staining for MKP-1 (A–C), Nrf2 (D–F), and HO-1 (G–I) from three representative lung adenoma tissues. Case 1, well-differentiated adenoma with weak positive staining for MKP-1 (A), Nrf2 (D), and HO-1 (G). Case 2, moderately-differentiated adenoma with moderate positive staining for MKP-1 (B), Nrf2 (E), and HO-1 (H). Case 3, poorly-differentiated adenoma with strong positive staining for MKP-1 (C), Nrf2 (F), and HO-1 (I). Original magnification ×400; scale bar, 50 μm.
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