Journal: British Journal of Cancer

Article Title: Spatial molecular analyses reveal key features associated with response to KN026 in advanced HER2-positive breast cancer

doi: 10.1038/s41416-025-03287-9

Figure Lengend Snippet: a Scatterplot illustrated prior gene expression profile including ERBB2, CDK12 and ESR1 stratified by response, where each point corresponds to an ROI. b Scatterplots comparing the normalized ERBB2 expression to CDK12 expression within PanCK+ tumor cells in tumor with OR and non-OR respectively. Two-sided test for association using Pearson’s correlation coefficient, r; p value shown on plots. c Data graphed as boxplots indicating differences in pre-treatment and post-treatment expression changes of ERBB2, CDK12 and ESR1 genes, as well as comparison between tumor with OR and non-OR. d Boxplot compared the changes of PI3K/AKT/mTOR and NOTCH signaling signatures within tumor cells following treatment in tumor with OR and non-OR. e Scatterplot comparison of mean DSP log2 negative-normalized expressions between ESR1 signaling and PI3K or NOTCH signaling within region of interest (ROI) in tumor with OR and non-OR. Two-sided test for association using Pearson’s correlation coefficient, r; p value shown on plots. f A panel of HER2-overexpressing ER-positive breast cancer cell line was treated with KN026 (100 µg/ml) for 48 h and whole-cell extracts were analyzed by western blotting with indicated antibodies. CCK8 assay evaluated the inhibition rate blocked by single or combined treatment of tamoxifen (1 µM) and KN026 (100 µg/ml). P values are based on One-way ANOVA test. g CCK8 assay evaluated the inhibition rate blocked by single or bispecific antibodies derived from constructed CDK12-amplified HER-2-overexpressed tumor cell lines. P values are based on One-way ANOVA test. h UMAP clustering showed the principle components between anti-HER2 antibodies. i Venn diagram illustrated that The KN026-treated tumor included ‘growth enhanced’ genes such as EGR1 programs from the tumor treated with trastuzumab and pertuzumab combinations, and 441. Overlap between the KN026 (purple), trastuzumab and pertuzumab (blue) and 441 (pink) genes ( n = 10, P = 1.29 × 10 − 4; two-sided hypergeometric). j PPI network illustrated the 77 differential expression genes shared between KN026 and 441. k Heatmap extracted the most significant KEGG pathways for each group. l Diagram showed the expression levels of CDK12, CALML5, and TFAP2B across different breast cancer subtypes using TCGA-BRCA data. m UMAP analysis characterized the expression levels of CDK12, CALML5, and TFAP2B in various breast cancer cell lines using the GSE298567 single-cell dataset and quantitated using violin plot ( n ) and Bubble diagram ( o ). p Illustrations depicted the mechanism of action of KN026 within breast cancer tumor cells. p value * p < 0.05, ** p < 0.01, *** p < 10^3, **** p < 10^4.

Article Snippet: Following 72 h after transfection, the cells were collected for western blotting as previously described (14) using a rabbit antibody against CDK12 (1:1000; ABclonal, Wuhan, China).

Techniques: Gene Expression, Expressing, Comparison, Western Blot, CCK-8 Assay, Inhibition, Derivative Assay, Construct, Amplification, Quantitative Proteomics

Journal: Molecular Biomedicine

Article Title: Malate targets pyruvate kinase M2 to promote colorectal cancer cell cycle arrest and tumor suppression

doi: 10.1186/s43556-025-00326-y

Figure Lengend Snippet: Malate mediates the inhibition of CRC cell proliferation and tumor growth by the CDC25A/CDK1 signaling pathway. a . Bubble chart shows the number of phosphorylation-modified peptides, phosphorylated sites, and proteins identified by 4D MS. b . Pathway analysis by the proteomic and phosphorylation proteomic analyses revealed cell cycle as the top protein-driven pathway. c . PPI network showing the proteins clustered in the cell cycle pathway and screening of hub genes using the MCC algorithm. d . The top 20 proteins were significantly associated with the pathways indicated in Fig. 4c. e . Malate treatment significantly promoted the phosphorylation levels of CDK1 in MC38 cells. After 48 h of malate treatment (7 mM), western blot was used to detect the protein levels of CDK1 and p-CDK1 in MC38 cells. f - g . The CDK phosphorylation inhibitor MK1775 eliminates the weakened effect of MC38 ( f ) and CT26 ( g ) cell proliferation mediated by malate treatment. h - i . The CDK phosphorylation inhibitor MK1775 eliminates the tumor growth delay effect mediated by malate treatment. 1 × 10 6 MC38 cells were subcutaneously injected into the groin of male C57 mice at 6–8 weeks of age. MK1775 (30 mg/kg) was administered orally on the 3rd, 5th and 7th days. Then oral administration of malate (200 mg/kg) was carried out daily for 5 days from the seventh day. On the fourteenth day, the mice were sacrificed to observe tumor progression. The tumor volum ( h ), the presentative images ( i , left) and tumor weight ( i , right). ( n = 3), scale bars, 1 cm. j . Malate did not affect the level of CDC25A mRNA. k . Malate treatment downregulated the protein level of CDC25A. After 48 h of malate treatment (7 mM), western blot was used to detect the protein levels of Wee1, CDC25A, CDK1 and p-CDK1 in MC38 cells. l . Malate cannot mediate the enhanced phosphorylation of CDK1 in MC38 cells with CDC25A knockdown (CDC25A KD ). After 48 h of malate treatment (7 mM), western blot was used to detect the protein levels of CDC25A, CDK1, and p-CDK1 in Ctrl or CDC25A KD MC38 cells. m . Malate promoted the degradation of CDC25A protein. Following 48-h malate treatment (7 mM) of MC38 cells, cycloheximide (CHX, 10 μg/mL) was administered to block protein synthesis. Cells were harvested at 0, 3, 6, 9, and 12 h post-CHX addition, and the protein level of CDC25A was detected by western blot. n . Malate-mediated degradation of CDC25A depends on the ubiquitin–proteasome pathway. o - p . Malate cannot inhibit the proliferation activity of CDC25A KD MC38 ( o ) or CT26 ( p ) cells. q - r . Malate cannot inhibit the progression of subcutaneous transplanted tumors of CDC25A KD MC38 cells. 1 × 10 6 Ctrl or CDC25A KD MC38 cells were subcutaneously injected into the groin of male C57 mice at 6–8 weeks of age. Then oral administration of malate (200 mg/kg) was carried out daily for 5 days from the seventh day. On the fourteenth day, the mice were sacrificed to observe tumor progression. The tumor volume ( q ), the presentative images ( r , left) and tumor weight ( r , right). ( n = 4), scale bars, 1 cm. All data represent the means ± s.e.ms. All data were analyzed with Student's t test. (*** P < 0.001, and n.s. not significant)

Article Snippet: Primary antibodies used in this experiment include P-CDK1, 2, 3, 5 (#ab133463-40ul, Abcam), β-actin (#66,009–1-IG-100UL, Proteintech), CDC25A (#55,031-AP-100ul, Proteintech), PKM2 (#60,268–1-1 g, Proteintech), BIP (#11,587–1-AP, Proteintech), Lamin B1 (#12,987–1-AP, Proteintech), WEE1 (#29,474–1-AP, Proteintech), CDK1 (#A11420, Abclonal), and p-CDK1 (#AP1350, Abclonal).

Techniques: Inhibition, Phospho-proteomics, Modification, Western Blot, Injection, Knockdown, Blocking Assay, Ubiquitin Proteomics, Activity Assay

Journal: Molecular Biomedicine

Article Title: Malate targets pyruvate kinase M2 to promote colorectal cancer cell cycle arrest and tumor suppression

doi: 10.1186/s43556-025-00326-y

Figure Lengend Snippet: Malate-PKM2 binding suppresses CRC via CDC25A-CDK1 axis. a . Malate treatment did not affect the protein levels of BiP in MC38 and CT26 cells. After 48 h of malate treatment (7 mM), western blot was used to detect the protein levels of BiP in MC38 or CT26 cells. b - c . The knockdown of BiP cannot block the inhibitory effect of malate-mediated proliferation in MC38 ( b ) or CT26 ( c ) cells. d . Venn Diagram. The potential binding protein of malate and the interaction protein of CDC25A were used to screen out the key protein PKM2. e . Drug affinity responsive target stability (DARTS). After the MC38 cell protein was lysed and incubated with malate at 37 °C for 30 min, different concentrations of protease were added for treatment, and the reaction was immediately terminated on ice. The protein level of PKM2 was detected by the western blot experiment. f . Microscale thermophoresis (MST) was used to investigate the binding of malate with PKM2. g . The protein levels of PKM2 and CDC25A were detected by Co-IP assay. h . Malate cannot mediate the reduction of CDC25A protein and the enhanced phosphorylation of CDK1 in PKM2 KD tumor cells. After 48 h of malate treatment (7 mM), western blot was used to detect the protein levels of PKM2, CDC25A, CDK1 and p-CDK1 in Ctrl or PKM2 KD MC38 cells. i . Malate did not affect the mRNA level of PKM2. j - k . Malate cannot inhibit the proliferation activity of PKM2 KD MC38 ( j ) or CT26 ( k ) cells. ( n = 5). l - q . Malate cannot inhibit the progression of subcutaneous transplanted tumors in PKM2 KD MC38 ( l - n ) or CT26 ( o - q ) cells. 1 × 10 6 ctrl or PKM2. KD MC38 or CT26 cells were subcutaneously injected into the groin of male C57/Babl/c mice at 6–8 weeks of age. Then oral administration of malate (200 mg/kg) was carried out daily for 5 days from the seventh day. On the fourteenth day, the mice were sacrificed to observe tumor progression. The tumor volume ( l and o ), the representative images ( m and p ) and tumor weight ( n and q ). ( n = 5 or 3), scale bars, 1 cm. All data represent the means ± s.e.ms. All data were analyzed with Student's t test. (* P < 0.05, *** P < 0.001, and n.s. not significant)

Article Snippet: Primary antibodies used in this experiment include P-CDK1, 2, 3, 5 (#ab133463-40ul, Abcam), β-actin (#66,009–1-IG-100UL, Proteintech), CDC25A (#55,031-AP-100ul, Proteintech), PKM2 (#60,268–1-1 g, Proteintech), BIP (#11,587–1-AP, Proteintech), Lamin B1 (#12,987–1-AP, Proteintech), WEE1 (#29,474–1-AP, Proteintech), CDK1 (#A11420, Abclonal), and p-CDK1 (#AP1350, Abclonal).

Techniques: Binding Assay, Western Blot, Knockdown, Blocking Assay, Incubation, Microscale Thermophoresis, Co-Immunoprecipitation Assay, Phospho-proteomics, Activity Assay, Injection

Journal: Molecular Biomedicine

Article Title: Malate targets pyruvate kinase M2 to promote colorectal cancer cell cycle arrest and tumor suppression

doi: 10.1186/s43556-025-00326-y

Figure Lengend Snippet: Malate allosterically binds PKM2 without perturbing its canonical activity. a . The molecule docking between malate and PKM2. b - d . Malate could not inhibit the proliferation activity of MC38 pretreated with Ser (200 μM, 6 h) by CCK8 ( b ) or BrdU ( c - d ) cell proliferation experiment. e . Malate cannot mediate the reduction of CDC25A protein and the enhanced phosphorylation of CDK1 after the pretreatment of Ser (200 μM, 6 h). f - g . Malate cannot inhibit the progression of subcutaneous transplanted tumors after the pretreatment of Ser. 1 × 10 6 MC38 cells were subcutaneously injected into the groin of male C57 mice at 6–8 weeks of age. L-Serine (5 mg/kg) was administered intratumorally on the 3rd, 5th and 7th days. Then oral administration of malate (200 mg/kg) was carried out daily for 5 days from the seventh day. On the fourteenth day, the mice were sacrificed to observe tumor progression. The tumor volume ( f ), the presentative images ( g , left) and tumor weight ( g , right). ( n = 4), scale bars, 1 cm. h - i . The PKM2 enzyme activity in tissues treated with malate. 1 × 10 6 MC38 ( h ) or CT26 ( i ) cells were subcutaneously injected into the groin of male C57 mice at 6–8 weeks of age. Then oral administration of malate (200 mg/kg) was carried out daily for 5 days from the seventh day. On the fourteenth day, the mice were sacrificed to detect tumor tissue PKM2 enzyme activity. ( n = 4). j . The lactic acid content in MC38 cells treated with malate (7 mM, 48 h). k . The lactic acid content in tumor tissues treated with malate. 1 × 10 6 MC38 cells were subcutaneously injected into the groin of male C57 mice at 6–8 weeks of age. Then oral administration of malate (200 mg/kg) was carried out daily for 5 days from the seventh day. On the fourteenth day, the mice were sacrificed to detect tumor tissue lactic acid content. ( n = 4). l - m . Malate treatment does not affect the expression of downstream genes regulated by PKM2. After 48 h of malate treatment (7 mM), the mRNA levels of Slc2a1, Hif1ɑ, LDHA, PDK1, and HK1 were detected in MC38 or CT26 cells. n . Malate treatment does not affect the nuclear translocation of PKM2. After 48 h of malate treatment, the protein levels of PKM2 in the cell nucleus and cytoplasm were detected separately. All data represent the means ± s.e.ms. All data were analyzed with Student's t test. (* P < 0.05, ** P < 0.01, *** P < 0.001, and n.s. not significant)

Article Snippet: Primary antibodies used in this experiment include P-CDK1, 2, 3, 5 (#ab133463-40ul, Abcam), β-actin (#66,009–1-IG-100UL, Proteintech), CDC25A (#55,031-AP-100ul, Proteintech), PKM2 (#60,268–1-1 g, Proteintech), BIP (#11,587–1-AP, Proteintech), Lamin B1 (#12,987–1-AP, Proteintech), WEE1 (#29,474–1-AP, Proteintech), CDK1 (#A11420, Abclonal), and p-CDK1 (#AP1350, Abclonal).

Techniques: Activity Assay, Phospho-proteomics, Injection, Expressing, Translocation Assay

Journal: Molecular Biomedicine

Article Title: Malate targets pyruvate kinase M2 to promote colorectal cancer cell cycle arrest and tumor suppression

doi: 10.1186/s43556-025-00326-y

Figure Lengend Snippet: Clinical sample validation of the clinical relevance of the malate/PKM2/CDC25A/CDK1 pathway. a . The expressions of MDH1, MDH2, FH, CDC25A, and p-CDK1 in CRC tissues and corresponding adjacent tissues were analyzed by immunohistochemical staining (n = 9 pairs). Each sample was sectioned continuously and stained. The magnifications of the picture from top to bottom are 200 times and 400 times, respectively. Scar bar: 1000 μm (upper), Scar bar: 200 μm (under). b - f . The immunostained tissues were scored by IRS. g - l . The correlations between MDH1, MDH2, FH, and CDC25A, p-CDK1 based on the IPS scores. m . Graphical model. CRC tissues exhibit reduced malic acid levels compared to normal tissues, impairing its interaction with PKM2. This disruption upregulates CDC25A protein level, attenuates CDK1 phosphorylation, and abrogates cell cycle arrest, thereby driving tumor progression. Exogenous malate supplementation restores this regulatory axis, exerting anti-tumor effects. Created with BioGDP.com. All data represent the means ± s.e.ms. All data were analyzed with Student's t test. (* P < 0.05, ** P < 0.01, and n.s. not significant)

Article Snippet: Primary antibodies used in this experiment include P-CDK1, 2, 3, 5 (#ab133463-40ul, Abcam), β-actin (#66,009–1-IG-100UL, Proteintech), CDC25A (#55,031-AP-100ul, Proteintech), PKM2 (#60,268–1-1 g, Proteintech), BIP (#11,587–1-AP, Proteintech), Lamin B1 (#12,987–1-AP, Proteintech), WEE1 (#29,474–1-AP, Proteintech), CDK1 (#A11420, Abclonal), and p-CDK1 (#AP1350, Abclonal).

Techniques: Biomarker Discovery, Immunohistochemical staining, Staining, Disruption, Phospho-proteomics