Nestin regulates cellular redox homeostasis in lung cancer through the Keap1–Nrf2 feedback loop

Abnormal cancer antioxidant capacity is considered as a potential mechanism of tumor malignancy. Modulation of oxidative stress status is emerging as an anti-cancer treatment. Our previous studies have found that Nestin-knockdown cells were more sensitive to oxidative stress in non-small cell lung cancer (NSCLC). However, the molecular mechanism by which Nestin protects cells from oxidative damage remains unclear. Here, we identify a feedback loop between Nestin and Nrf2 maintaining the redox homeostasis. Mechanistically, the ESGE motif of Nestin interacts with the Kelch domain of Keap1 and competes with Nrf2 for Keap1 binding, leading to Nrf2 escaping from Keap1-mediated degradation, subsequently promoting antioxidant enzyme generation. Interestingly, we also map that the antioxidant response elements (AREs) in the Nestin promoter are responsible for its induction via Nrf2. Taken together, our results indicate that the Nestin–Keap1–Nrf2 axis regulates cellular redox homeostasis and confers oxidative stress resistance in NSCLC.

total antioxidant activity (T-AOC) was assessed.
(g and h) qPCR analysis showing that overexpression of Myc-Nestin in A549 and H1299 cells affects the expression levels of several antioxidation-related genes compared to those seen in control cells.
(i) Analysis of GSH levels in NSCLC cells.
(j) SOD activity in NSCLC cells was examined with a SOD assay kit.
(k) Analysis of CAT levels in NSCLC cells with or without Myc-Nestin.
Data are presented as the means ± SD of three independent experiments. * P < 0.05, **P < 0.01 and ***P < 0.001, Student's t test. Source data are provided as a Source Data file. (e) Immunofluorescence was used to detect the expression of Nestin, GCLM and HO-1 in human xenograft tumors of mice treated with/without Dox. H1299 cells were labeled with anti-GCLM/HO-1 (red), anti-Nestin (green) and DAPI (blue). Scale bar: 50 μm.
(f) Immunofluorescence was used to detect ROS levels in human xenograft tumors of mice treated with/without Dox. NSCLC cells were labeled with DHE (red), anti-Nestin (green) and DAPI (blue). Scale bar: 50 μm.
Error bars represent the SD of data obtained from three independent experiments. * P < 0.05, **P < 0.01 and ***P < 0.001, Student's t test. Source data are provided as a Source and HMOX1 (d), were reduced in Nestin-knockdown H1299 cells. H1299 cells were treated with 100 μM tBHQ or 20 μM SF for 16 h, and qPCR was used to measure the relative amounts of the mRNAs for GCLM, NQO1 and HMOX1.
(e and f) Overexpression of Nestin is not notably affected by depletion of Nrf2 in A549 cells. Nrf2-knockdown NSCLC cells were transfected with Vector or Myc-Nestin. The results carried out via detecting the Nrf2 downstream genes expression by qPCR.
* P < 0.05, **P < 0.01 and ***P < 0.001, Student's t test. Source data are provided as a Source Data file.  (h) Flow cytometric analysis with Annexin V-FITC and propidium iodide (PI) was used to detect apoptosis and necrosis.
(i) Statistical analysis of the total apoptosis rates in NSCLC cells.
Error bars represent the SD of data obtained from three independent experiments. * P < 0.05, **P < 0.01 and ***P < 0.001. N.S. represents no significant, Student's t test. Source data are provided as a Source Data file. (a and b) A549 and H1299 cells were treated with PBS or H2O2, and chromatin immunoprecipitation was performed using Nrf2-specific antibodies. DNA isolated from the precipitated materials was analyzed using qPCR with the indicated primers. The ARE-specific signals from Nrf2-precipitated DNA were normalized to those from IgG-precipitated DNA.
The data shown are means ± SD of triplicate wells. * P < 0.05 and **P < 0.01, Student's t test. Source data are provided as a Source Data file.  (d) Statistical analysis of the total apoptosis rate in NSCLC cells.