Fig 2: Sulfenylated AHR–regulated glycogenolysis promotes drug resistance in vivo.(A) Schematic of experimental design. (B–D) Tumor-bearing mice were administrated with DDP. The tumor growth (B) and mouse survival (C) were monitored. ROS and NADPH/NADP+ (D) in isolated tumor cells were analyzed. (E) Schematic of experimental design for mouse treatment. (F) DDP-treated tumor-bearing mice were administrated with 6-AN. ROS and NADPH/NADP+ were analyzed. (G) Immunohistochemical staining of p-PYGL from the sections of tumor tissues in tumor-bearing mice. Scale bar, 50 μm. (H and I) DDP-treated tumor-bearing mice were administrated with GPI, tumor growth was monitored, and ROS or NADPH/NADP+ were analyzed. (J) Immunohistochemical staining of AHR from the sections of tumor tissues in tumor-bearing mice. Scale bar, 50 μm. (K) Isolated tumor cells were labeled with dimedone. AHR (green) and PTG (cyan) were observed under super-resolution microscope. The arrows indicate the colocation of the sulfenylated AHR and PTG. Scale bars, 10 μm. (L and M) DDP-treated tumor-bearing mice were administrated with SR1. ROS and NADPH/NADP+ were analyzed and tumor growth was monitored. (N) AHR–/– and AHR(C300A)- or AHR(C300)-overexpressing (OE) DRCs were inoculated into NSG mice. Tumor growth of treated tumor-bearing mice was monitored. (O) DDP-treated tumor-bearing mice were administrated with NAC 5 times or pretreated with NAC for 3 days before being administrated with DDP. Tumor size was presented photographically (top) or by weight (bottom). Scale bars, 1 cm. (P and Q) DDP-resistant MMTV-PyMT mice were administrated with DDP 3 times. ROS levels and NADPH/NADP+ in isolated tumor cells were analyzed. AHR (green) and PTG (cyan) were observed. The arrows indicate the colocation of the sulfenylated AHR and PTG. Scale bars, 10 μm. A–M, n = 6 mice; N–Q, n = 5 mice. All error bars are mean ± SD. P values were calculated by 1-way ANOVA followed by Bonferroni’s test (B, D, F, H, I, and L–P), and log-rank test (C); *P < 0.05, **P < 0.01, ***P < 0.001. qod, every other day.

Fig 3: Cysteine sulfenylation licenses AHR to bind to glycogen particles.(A) Immunoblot of immunoprecipitations of STBD1 or HSP90 in lysates from MCF-7 or MCF-7/DDP cells treated with DDP for 24 hours. (B) Strategy for detecting sulfenic acid modification of AHR with dimedone. LC-MS/MS analysis of dimedone-labeled AHR. Analysis of the y-ions indicates the formation of dimedone adduct (+138.07 Da). (C) MCF-7/DDP or A549/5-Fu cells were treated with DDP or 5-Fu for 24 hours. The location of AHR (green), dimedone (red), and STBD1 (cyan) was observed under super-resolution microscope. The arrows indicate the colocation of the sulfenylated AHR and STBD1. Scale bars, 10 μm. (D) Separated glycogen-enriched pellets from MCF-7/DDP or A549/5-Fu cells treated with DDP or 5-Fu for 24 hours were resuspended in IP buffer, and immunoblot with dimedone of immunoprecipitations of AHR was analyzed. (E) Purified Flag-tagged (Flag-AHR) protein was treated with different doses of H2O2 for 30 minutes. Sulfenic acid modification of dimedone-labeled AHR was analyzed by Western blot. (F) Purified Flag-AHR protein was treated with different doses of H2O2 for 30 minutes. The binding between modified AHR and HSP90 was measured by biolayer interferometry (BLI). (G) HEK293T cells transfected with the indicated combinations of Flag-HSP90, Myc-AHR, or Myc-AHR(C300A) were treated with H2O2 for 30 minutes. Immunoprecipitations of Flag or STBD1 were analyzed by Western blot.

Fig 4: DRCs use AHR to promote glycogenolysis.(A) MCF-7 or MCF-7/DDP cells were treated with DDP for 24 hours. Cell lysates were immunoprecipitated with anti-STBD1 for MS. Identified proteins are listed. (B) Immunoblot of immunoprecipitations of STBD1 in lysates from cells treated with DDP or 5-Fu for 24 hours. (C) Cells were treated with DDP or 5-Fu for 24 hours. The location of AHR (green) and STBD1 (red) was observed under confocal microscope. Scale bar, 10 μm. (D) MCF-7/DDP or A549/5-Fu cells transduced with si-NC or si-AHR were treated with DDP or 5-Fu for 24 hours. Phosphorylated (phospho-) PYGL or PYGL was analyzed by Western blot. (E) MCF-7/DDP or A549/5-Fu cells cultured in 13C-glucose transduced with si-NC or si-AHR were switched to 12C-glucose for 4 hours of drug treatment, and 13C-labeled R5P was detected by LC-MS/MS. (F and G) MCF-7/DDP or A549/5-Fu cells transduced with si-NC or si-AHR were treated with DDP or 5-Fu for 24 hours. NADPH/NADP+ (F) and ROS levels (G) were analyzed. (H) MCF-7/DDP or A549/5-Fu cells cultured in 13C-glucose for 10 days were transduced with si-NC or si-AHR, then treated with DDP or 5-Fu for 8 hours, followed by treatment with hydrochloric acid, leading to the degradation of polymer glycogen into monomer glucose. The amount of released 13C-labeled glucose (m+6) was determined by LC-MS/MS. The effect of blocked glycogen degradation by AHR knockdown is evaluated by the amount of m+6 glucose, with the formula ([si-AHR1% + si-AHR2%]/2) – si-NC%. All error bars are mean ± SD. P values were calculated by 1-way ANOVA followed by Bonferroni’s test (E–H); n = 3; **P < 0.01, ***P < 0.001.

Fig 5: AHR glycogenolysis occurs in patients with chemoresistant cancer.(A–C) Overall survival on the basis of AHR level in people with breast cancer (n = 1,669) or lung cancer (n = 38) (A), PYGL level in people with breast cancer (n = 1,669) or lung cancer (n = 555) (B), and G6PD level in people with breast cancer (n = 1,669) or lung cancer (n = 555) (C). (D and E) The tissue sections from patients with breast cancer including responders or nonresponders before and after chemotherapy were immunohistochemically stained with anti-AHR antibody (D) and anti–p-PYGL antibody (E). Scale bars, 50 μm. (n = 6 per group.) (F) Isolated primary tumor cells from fresh breast cancer tissues including responders and nonresponders after chemotherapy were treated with dimedone (5 mM) for 30 minutes. The location of AHR (green), dimedone (red), and PTG (cyan) in the isolated tumor cells of tumor tissues was observed under super-resolution microscope. Scale bars, 10 μm. (n = 6 per group.) (G–I) ROS levels (G), the ratio of NADPH/NADP+ (H), and R5P and S7P (I) in isolated primary tumor cells from fresh breast cancer tissues including responders and nonresponders after chemotherapy were analyzed (n = 6 per group). All error bars are mean ± SD. P values were calculated by 1-way ANOVA followed by Bonferroni’s test (D and E), 2-tailed unpaired Student’s t test (G–I), and log-rank test (A–C). **P < 0.01, ***P < 0.001.