Mitochondrial GRIM-19 deficiency facilitates gastric cancer metastasis through oncogenic ROS-NRF2-HO-1 axis via a NRF2-HO-1 loop

Abstract

Background NRF2, a prime target of cellular defense against oxidative stress, has shown a dark side profile in cancer progression. GRIM-19, an essential subunit of the mitochondrial MRC complex I, was recently identified as a suppressive role in tumorigenesis of human gastric cancer (GC). However, little information is available on the role of GRIM-19 and its cross-talk with NRF2 in GC metastasis. Methods Online GC database was used to investigate DNA methylation and survival outcomes of GRIM-19. CRISPR/Cas9 lentivirus-mediated gene editing, metastasis mice models and pharmacological intervention were applied to investigate the role of GRIM-19 deficiency in GC metastasis. Quantitative RT-PCR, FACS, Western blot, IHC, IF and reporter gene assay were performed to explore underlying mechanisms. Results Low GRIM-19 is correlated with poor survival outcome of GC patients while DNA hypermethylation is associated with GRIM-19 downregulation. GRIM-19 deficiency facilitates GC metastasis and triggers aberrant oxidative stress as well as ROS-dependent NRF2-HO-1 activation. Experimental interventions of specific ROS, NRF2 or HO-1 inhibitor significantly abrogate GRIM-19 deficiency-driven GC metastasis in vitro and in vivo. Moreover, HO-1 inhibition not only reverses GRIM-19 deficiency-driven NRF2 activation, but also feedback blocks NRF2 activator-induced NRF2 signaling, resulting in decreased metastasis-associated genes. Conclusions Our data suggest that GRIM-19 deficiency accelerates GC metastasis through the oncogenic ROS-NRF2-HO-1 axis via a positive-feedback NRF2-HO-1 loop. Therefore, this study not only offers novel insights into the role of oncogenic NRF2 in tumor progression, but also provides new strategies to alleviate the dark side of NRF2 by targeting HO-1.

Antidiabetic Agent DPP-4i Facilitates Murine Breast Cancer Metastasis by Oncogenic ROS-NRF2-HO-1 Axis via a Positive NRF2-HO-1 Feedback Loop

“…Cell migration and cell invasion assays were performed in 24well non-coated or Matrigel-coated Transwell chambers (8-µm pore size, Corning, NY, USA) as described previously (12)(13)(14). Briefly, 4.5 × 10 4 cells for cell migration or 1 × 10 5 cells for cell invasion were plated in the upper chamber with 200 ml of serumfree medium, and 800 ml medium supplemented with 10% FBS was used as a chemoattractant in the bottom chamber.…”

“…RNA isolation and qRT-PCR were performed as described previously (12)(13)(14)(15)(16)(17). Total RNA from harvested cells was isolated using Tripure Isolation Reagent (Roche, Mannheim, Germany).…”

“…4T1 cells were treated with indicated regents and then subject to Western blotting analysis using RIPA buffer (Beyotime, Haimen, China) as described previously (12)(13)(14)(15)(16). Protein lysates after SDS-PAGE were blotted onto PVDF membranes, blocked in QuickBlock ™ Blocking Buffer for Western Blot (Beyotime, Haimen, China) and followed by primary antibody incubation.…”

“…H&E and IHC staining were performed as described previously (6,(12)(13)(14)(15)(16). Briefly, lung or liver tissues were fixed with 10% buffered formalin and embedded in paraffin.…”

Upregulation of Linc-ROR Promotes the Proliferation, Migration, and Invasion of Gastric Cancer Cells Through miR-212-3p/FGF7 Axis

“…After transfection with miR-212-3p mimics or antagomir for 24 hours, cell migratory and invasive capacities were analyzed using transwell assay (BD Sciences) as described previously. 25 Cell suspensions (5×10 4 cells/mL) were prepared using a serum-free medium. Into each well of the upper chamber, 100 μL of the cell suspension was added; the lower chamber contained 600 μL of the culture medium supplemented with 10% FBS.…”