Effects of KEAP1 Silencing on the Regulation of NRF2 Activity in Neuroendocrine Lung Tumors

Abstract

Background: The KEAP1/NRF2 pathway has been widely investigated in tumors since it was implicated in cancer cells survival and therapies resistance. In lung tumors the deregulation of this pathway is mainly related to point mutations of KEAP1 and NFE2L2 genes and KEAP1 promoter hypermethylation, but these two genes have been rarely investigated in low/intermediate grade neuroendocrine tumors of the lung.

Methods: The effects of KEAP1 silencing on NRF2 activity was investigated in H720 and H727 carcinoid cell lines and results were compared with those obtained by molecular profiling of KEAP1 and NFE2L2 in a collection of 47 lung carcinoids. The correlation between methylation and transcript levels was assessed by 5-aza-dC treatment.

Results: We demonstrated that in carcinoid cell lines, the KEAP1 silencing induces an upregulation of NRF2 and some of its targets and that there is a direct correlation between KEAP1 methylation and its mRNA levels. A KEAP1 hypermethylation and Loss of Heterozygosity at KEAP1 gene locus was also observed in nearly half of lung carcinoids.

Conclusions: This is the first study that has described the effects of KEAP1 silencing on the regulation of NRF2 activity in lung carcinoids cells. The epigenetic deregulation of the KEAP1/NRF2 by a KEAP1 promoter hypermethylation system appears to be a frequent event in lung carcinoids.

Keywords: KEAP1; Lung Carcinoid; NRF2; methylation; mutation; outcome.

Figure 1

(A) Representative Western Blots analysis showing expression levels of KEAP1, NRF2, AKR1C1 and TXNRD1 in H720 and 727 cell lines after KEAP1 inhibition by specific KEAP1 siRNA. Scrambled siRNA was performed as the control. (B) Histograms show the expression levels of the proteins normalized to actin (N = 4). No overexposure was done. The grouping of gels/blots were cropped from different parts of the same gels. * p < 0.05, ** p < 0.001, *** p < 0.0001 t-test.

Figure 2

(A) Expression level analysis (± standard error mean) of the KEAP1 gene in H720 and H727 determined by RT-qPCR. The relative quantification was expressed as the ratio marker (KEAP1/RPLPO). (B) Methylation levels (± standard error mean) of KEAP1 in H720 and H727 determined by qMSP (N = 4). * p < 0.05, *** p < 0.0001 t-test.

Figure 3

(A) Changes in KEAP1 mRNA transcript levels in the H720 cell line before (Untreated) and after treatment with 5-aza-dC at 24 h and 48h. Error bars indicate the standard deviation of three different experiments. * p < 0.01, t-test. (B) Changes in KEAP1 promoter methylation levels in the H720 cell line by quantitative methylation real-time PCR before (CTRL) and after treatment with 5-aza-dC at 48 and 72 h. Error bars indicate the standard deviation of three different experiments. * p < 0.05, t-test.

Figure 3

(A) Changes in KEAP1 mRNA transcript levels in the H720 cell line before (Untreated) and after treatment with 5-aza-dC at 24 h and 48h. Error bars indicate the standard deviation of three different experiments. * p < 0.01, t-test. (B) Changes in KEAP1 promoter methylation levels in the H720 cell line by quantitative methylation real-time PCR before (CTRL) and after treatment with 5-aza-dC at 48 and 72 h. Error bars indicate the standard deviation of three different experiments. * p < 0.05, t-test.

Figure 4

Immunohistochemistry analysis showing KEAP1, NRF2, TXNRD1 and NQO1 proteins expression in one typical (unmethylated, A–D) and one atypical (methylated, E–H) lung tissue from two different carcinoid affected patients. 20× and 40× magnification. A strong KEAP1 cytoplasmatic staining was observed in a typical unmethylated lung carcinoid tissue with a mild nuclear NRF2 and cytoplasmatic TXNRD1 and NQO1 staining, whereas an inverse protein staining pattern was observed in an atypical methylated lung carcinoid tissue.