Fig 2: Blocking AURKA stalls the proliferation of GBM by reducing glycolysis in a c-Myc dependent manner.a Volcano plot of reverse-phase-protein-array (RPPA) data shows a reduced expression of c-Myc (red dot) in SF188 GBM cells treated with 100 nM alisertib for 24 h (n = 3 independent samples). FC: fold change. b Protein capillary electrophoresis of SF188 and GBM22 cells treated with the indicated concentrations of alisertib for 24 h. Vinculin is used as a loading control. c SF188 was transfected with non-targeting (siNT) or AURKA specific siRNA (siAURKA) or transduced with shARKA (shRNA) or sgAURKA (sgRNA) and the whole-cell protein lysates were subjected to protein capillary electrophoresis. d SF188 and GBM22 cells were transfected with non-targeting (siNT) or two specific siRNAs targeting Myc, treated with increasing concentrations of alisertib for 72 h, and cellular viability was analyzed (n = 4 independent samples). IC50 in µM range. e SF188 and GBM22 cells were infected with empty vector or c-Myc adenovirus and treated with increasing concentration of alisertib for 72 h, and cellular viability was analyzed (n = 4 independent samples). IC50 in µM range. f SF188 and GBM22 cell lysates were immunoprecipitated with IgG or c-Myc antibody and analyzed by standard western blot for the indicated antibodies. Input: cell lysate loading control. IgG: negative control. g SF188 cells were treated with DMSO or alisertib in the presence or absence of 5 µM MG132 and the whole-cell lysates were subjected to protein capillary electrophoresis for the indicated proteins. For h, i SF188 cells were treated with DMSO or alisertib in the presence or absence of 10 µg/mL cycloheximide (CHX) and the whole-cell lysates were subjected to protein capillary electrophoresis. Quantification of c-Myc protein level is shown in (i) (n = 3 independent samples). For j, k protein capillary electrophoresis for the indicated proteins of SF188 and GBM22 cells treated with DMSO or alisertib for different time points. Quantification of protein level is shown in (k) (n = 2 independent samples). l SF188 cells were transfected with c-Myc-WT or c-Myc mutant (T58A), treated with the indicated concentrations of alisertib for 24 h, and analyzed by protein capillary electrophoresis for the indicated proteins. m GBM22-Myc-WT or GBM22-T58A-Myc cells were implanted in the right striatum of nude mice. Two groups were randomly assigned: vehicle and alisertib after seven days of the implantation. Mice were treated three times per week and animal survival is provided (Kaplan-Meier-curve): GBM22-T58A-Myc-vehicle: 34d, GBM22-T58A-Myc-alisertib: 27d; GBM22-Myc-OE-vehicle: 38d, GBM22-Myc-OE-alisertib: 50d. The log-rank test was used to assess statistical significance (n = 5 independent samples) (*p = 0.0127, n.s not significant). n SF188 and GBM22 cells were transfected with non-targeting siNT or siAURKA in the presence or absence of 2 µM CHIR-908014 (CHIR) for 24 h and were subjected to protein capillary electrophoresis for the indicated proteins. o SF188 and GBM22 cells were transfected with HA-EV, HA-Aurora A-WT, HA-Aurora A-D274N and were subjected to protein capillary electrophoresis for the indicated proteins. Statistical significance was assessed by two-tailed student’s t-test in (m). Data are shown as mean ± SD in (d, e, i, k). Source data are provided as a Source Data file.

Fig 3: High expression of MYC, CCAAT/enhancer-binding protein delta (CEBPD) and HK2 along with low FBXW7 and hsa-miR-429 expression predicts adverse features and poor patient outcomes in urinary bladder urothelial carcinoma (UBUC) and upper urinary tract urothelial cancer (UTUC). (A) IHC and in situ hybridization showed that MYC amplification; high expression of MYC, CEBPD, and HK2; and low expression of FBXW7 and hsa-miR-429 were strongly relevant to UBUC patients (n = 295) with high tumour stage. (B–C) Moreover, Kaplan-Meier survival analysis indicated that both MYC amplification and high MYC expression are significant survival determinants in UBUC (n = 295; B1 and B2) and UTUC (n = 340; C1 and C2), while the survival impacts of MYC expression can be further enriched by the coexpression of CEBPD. (B3 and C3), suggesting potential synergistic effects of MYC and CEBPD in the promotion of UC progression. In addition, low expression of miR-429 and high expression of HK2 also confer a poor prognosis in terms of disease-specific survival in UBUC (n = 295; B4 and B5) and UTUC (n = 340; C4 and C5) patients. The UBUC and UTUC cohorts were taken from the biobank of Chi Mei Medical Center that collected specimens after an operation with curative intent between January 1996 and May 2004 as previously described. 22 This study was approved by the institutional review board of Chi Mei Medical Center (IRB10207-001). For immunohistochemistry, one representative image was shown upper urinary tract urothelial cancer (UTUC)

Fig 4: Diabetes mellitus (DM) exacerbates CCAAT/enhancer-binding protein delta (CEBPD)-driven tumour aggressiveness. (A) Analysis of the National Health Insurance Research Database (NHIRD) showed that urinary bladder urothelial carcinoma (UBUC) (A1, n = 8436, p < .001) and upper urinary tract urothelial cancer (UTUC) (A5, n = 3232, p = .003) patients with concomitant DM had a higher death rate than non-DM patients. However, unlike the results for the NHIRD, which includes a large number of patients, concomitant DM only displayed marginal significance for the prediction of poor disease-specific survival (DSS) in UBUC (A2, n = 295, p = .0547) and UTUC (A6, n = 340, p = .0867) in our cohort, which includes few cases, indicating that the significance of DM in patients’ outcome might be mild. Interestingly, for those cases with high CEBPD expression in our cohort, the presence of DM conferred a significantly worse prognosis in UBUC (A3, n = 88, p = .0046) and UTUC (A7, n = 89, p = .0187). Conversely, tumours harbouring high CEBPD expression were even more aggressive in DM patients in both the UBUC (A4, n = 55, p = .0001) and UTUC (A8, n = 64, p < .0001) cohorts. The aforementioned evidence revealed that CEBPD-mediated aggressiveness in UC can be exacerbated by concomitant hyperglycemic disorder, a symptom that characterizes diabetes, probably because high-glucose conditions help to reinforce CEBPD-mediated glycolysis in cancer cells. (B) Experiments on a BFTC909-derived xenograft model (n = 8 for each group) further reinforced that the effects of CEBPD on promoting tumour growth could be exacerbated by concomitant hyperglycemia. First, compared with the mock conditions, CEBPD overexpression promoted the growth of xenografted tumours in SCID/beige mice without high-fat diet-induced DM. Accelerated tumour growth driven by CEBPD overexpression was exacerbated in mice with high-fat-diet-induced DM compared with those with CEBPD overexpression alone or induced DM alone (B1). The CEBPD-induced and DM-induced effects on tumour aggressiveness and the DM-induced exacerbation of CEBPD-driven tumour growth were all statistically significant (B2). (C) IHC analysis of xenografted samples at day 25 post-injection showed that pAKT1, pMTOR, pRPS6, pEIF4EBP, MKI67, MYC and HK2 were highly expressed and that hsa-miR-429 were downregulated in the CEBPD-overexpressing and/or DM-induced groups compared to the control group. Of note, the upregulation of MYC and HK2 and downregulation of hsa-miR-429 were more prominent in the CEBPD-overexpressing group than in the mock group, regardless of DM induction. This suggests that the CEBPD-mediated effects on glycolysis might be regardless of hyperglycemia or normoglycemia conditions. For immunohistochemistry, one representative image was shown. Data are shown as the mean ± SEM. Statistical significance: * p < .05

Fig 5: Blocking AURKA stalls GBM growth by reducing glycolysis.a SF188 and GBM22 cells were cultured in galactose or glucose media for a week, treated with increasing concentrations of alisertib for 72 h, and cellular viability was analyzed (n = 4 independent samples). b SF188 GBM cells either transduced with a non-targeting (sgNT) or two different AURKA (sgAURKA) sgRNAs were cultured in galactose or glucose media for a week and treated with increasing concentration of alisertib. Cellular growth over time was determined (n = 4 independent samples). For c, d SF188 and GBM22 cells were treated with DMSO or alisertib and analyzed in the context of a glycolysis stress assay on a Seahorse XFe24 extracellular flux analyzer. Extracellular acidification rate (ECAR) is recorded at baseline, after injection of Glucose (G), Oligomycin (OM), and 2-DG. Quantification of glycolysis is shown in (d) (n = 5 independent samples). e Real-time PCR analysis of the mRNA level of genes related to glycolysis in SF188 and GBM22 cells treated with DMSO or alisertib for 24 h (n = 8 in SF188 + DMSO and GBM22 + DMSO, n = 4 in SF188 + Ali 0.1 µM and GBM22 + Ali 1 µM, independent samples). (HK2: SF188 ***p = 0.0002, GBM22: ***p = 0.0003; **p = 0.0082, ****p < 0.0001). 18S: internal control. FC: fold change. f SF188 and GBM22 cells were treated with DMSO or alisertib for 24 h and the whole-cell protein lysates were subjected to protein capillary electrophoresis. Vinculin is used as a loading control. g SF188 cells were transfected with non-targeting siRNA or specific AURKA siRNAs (single or pool) and the whole-cell lysates were analyzed by protein capillary electrophoresis for the indicated proteins. h Real-time PCR analysis of the mRNA level of genes related to glycolysis in SF188 transfected with non-targeting siRNA or specific siRNA targeting AURKA (n = 4 independent samples) (*p = 0.0179, ***p = 0.0001). i Shown are the ATP levels measured by polar LC/MS of SF188 cells treated with DMSO or 100 nM alisertib (n = 5 independent samples) (*p = 0.0221). For j, k SF188 and GBM22 cells were transfected with non-targeting or specific siRNA targeting AURKA and were analyzed in the context of a glycolysis stress assay on a Seahorse XFe24 extracellular flux analyzer. The graphs show glycolysis level in (j) (SF188: n = 2 in siNT, n = 3 in siAURKA; GBM22: n = 3 independent samples) (**p = 0.0032) or OCR/ECAR levels in (k) (SF188: n = 3; GBM22: n = 3 in siNT, n = 2 in siAURKA independent samples) (***p = 0.0001). l SF188 cells were transfected with c-Myc-WT and c-Myc mutant (T58A), treated with increasing concentrations of alisertib for 24 h, and the whole-cell lysates were analyzed by protein capillary electrophoresis. m SF188 cells were transfected with c-Myc-WT and c-Myc mutant (T58A), treated with 50 nM alisertib for 24 h, and analyzed on a Seahorse XFe24 extracellular flux analyzer. Shown is the quantification of ECAR level (n = 5 independent samples) (**p = 0.0073, n.s not significant). n SF188 and GBM22 cells were treated with alisertib for 24 h and were subjected to CHIP with an IgG as a negative control or a c-Myc specific antibody. The HK2 region was amplified by PCR (n = 3 independent samples). Statistical significance was assessed by a two-tailed student’s t-test. Data are shown as mean ± SD in (a-e, h-k, m, n). Source data are provided as a Source Data file.