Fig 2: HK2 together with SENP1 upregulation associates poor outcome and worse chemotherapy response in prostate cancer patients.a Representative images of immunohistochemistry staining of HK2 and SENP1 in Renji prostate cancer tissue microarray. Scores were calculated by intensity and percentage of stained cells. Scale bar, 600 μm. b Kaplan–Meier survival analysis in HK2 high subgroup of prostate cancer biochemical progression-free survival (BPFS) defined as SENP1 low or high expression, using Renji tissue microarray cohort. Statistical difference was determined by a two-sided log-rank test. NS not significant. c Relationship between SENP1 staining and Gleason Score in HK2 low or high subgroup, using Renji tissue microarray cohort. Gleason Score was divided in low ≤6, medium = 7, and high 8–10. The distribution of high Gleason Score increased in SENP1 and HK2 both high group. Statistical significance was measured by the chi-square test. NS not significant. d The percentage of biochemical recurrence <1 or ≥1 year of docetaxel-based neoadjuvant chemohormonal therapy prostate cancer patients (n = 48 samples), stratified by HK2 and SENP1 expression. Statistical significance was measured by the chi-square test. NS not significant. e Schematic diagrams illustrating the critical role of HK2 SUMOylation in prostate cancer cell docetaxel treatment. Source data are provided as a Source Data file.

Fig 3: Administering the glycolysis inhibitor metformin based on HK2 circadian rhythm improves trastuzumab efficacy. A and B, Western blot analysis of PER1, AMPK, and p-AMPK proteins in TR cells treated with MET with or without AMPK knockdown (A) or MG132 treatment (B). C, NCI-N87TR cells were subcutaneously injected in nude mice under a 12:12 h light/dark cycle with the lights on from ZT0 to ZT12. Mice were treated with PBS, trastuzumab (TRA; 10 mg/kg), metformin (MET; 250 mg/kg), or TRA combined with MET at ZT6 or ZT18. Western blot analysis was performed to detect PER1, HK2, AMPK, and p-AMPK in PBS and MET groups at ZT6 or ZT18. D, Nude mice were killed at 4 hours intervals across the 24 hours light/dark cycle for two consecutive days. qPCR analysis of relative mRNA levels of clock genes BMAL1, CLOCK, PER1, and PER2 in PBS and MET (250 mg/kg) groups are shown (n = 3). E and F, Tumor volumes (E) and weight (F) of nude mice were calculated (n = 5). One-way ANOVA was used to calculate P values. G, Representative IHC images of Ki67, cleaved-caspase-3, PER1, and HK2 performed on serial sections of subcutaneous tumors of xenograft nude mice. Scale bar, 140 µm. Data are graphed as the mean ± SD; *, P < 0.05; ***, P < 0.001; ns, nonsignificant, P > 0.05.

Fig 4: FGF7 promotes mitochondrial localization of HXK2 to protect against superoxide injury. HXK2VBD, which inhibits mitochondrial localization of HXK2, were pretreated for 1 h before OGD treatment and FGF7 administration in NRCMs. (A) Representative immunofluorescence analysis of HXK2 (green) in NRCMs. The COX IV immunostaining (red) highlights mitochondria, and nuclei were stained with DAPI (blue), scale bars: 10 µm. (B) Mitochondrial was isolated to detect the HXK2 protein levels in NRCMs. COX IV and ß-Actin were served as loading controls for mitochondrial. (C) White lines in merged images (A) indicate the area where the distances between HXK2, COX IV, and DAPI were analyzed using ImageJ. (D) Mitochondrial membrane potential was detected by TMRM ?uorescence staining, scale bars: 25 µm. (E) mtROS of NRCMs was detected by MitoSOX staining assay, scale bars: 50 µm. (F–G) Representative images of (F) DHE staining and (G) TUNEL staining of NRCMs. Scale bar: F, 100 µm; G, 50 µm n = 3 in per group. mtROS: mitochondrial reactive oxygen species. All values are presented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig 5: Schematic of the mechanism of action of ETV4 in promoting breast cancer stemness.ETV4 promotes BCSC maintenance, on the one hand, by promoting transcription of HK2 and LDHA to augment glycolytic flux, and onthe other, by transcriptionally activating CXCR4 expression to stimulate SHH/GLI1 signaling cascade.