Fig 2: KMT5A expression was significantly correlated with activated MARK4/YAP signaling in triple-negative breast cancer.a Representative images of KMT5A, SNIP1K301me1, and MARK4 in 100 clinical TNBC metastatic lymph node tissues. Scale bars, 50 µm. b Correlation of expression levels between KMT5A, SNIP1K301me1, and MARK4 in (a). c Prognosis comparison of breast cancer patients with KMT5A/SNIP1K301me1 or KMT5A/MARK4 ectopic differential expression using Kaplan–Meier survival analysis. d Representative immunoblots showing KMT5A, SNIP1K301me1, and MARK4 expression in normal breast specimen (N) and paired tumor tissues (T) (n = 3). e Correlation between TNBC stages and IHC scores of KMT5A and SNIP1K301me1. f Representative IHC staining of KMT5A, SNIP1K301me1, and MARK4 in primary TNBC and matched metastatic lymph nodes (n = 100 paired samples). Scale bars: 50 µm. g A working model of KMT5A-promoted Hippo/YAP signaling pathway modification and triple-negative breast cancer metastasis through the SNIP1 methylation status. KMT5A methylated SNIP1 at K301, which released KAT2A for activated histone acetyltransferase (HAT) and recruited by c-MYC, leading to enhanced KAT2A anchoring onto the MARK4 promoter and MARK4 transcription, ultimately activating the Hippo/YAP signaling pathway. Data information: In (b), Statistical analysis was performed by chi-square test. In (c), by log-rank test. In (e), by two-tailed t-test. *P < 0.05, ***P < 0.001. Data are represented as mean ± SEM. Panel (d) shows one experiment representative of three independent experiments with similar results.

Fig 3: KMT5A-mediated SNIP1 K301 methylation activates Hippo/YAP signaling.a Heatmap of RNA-Seq analysis of differentially expressed genes downregulated by SNIP1-K301R (SNIP1K301R) (twofold change and FDR < 0.05). SNIP1 sgRNA-resistant Flag-SNIP1WT and SNIP1K301R mutant were re-expressed in sgSNIP1/MDA-MB-231 cells. b Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis indicates that the genes downregulated by the SNIP1K301R mutant were predominantly associated with the Hippo signaling pathway. c Venn diagram shows YAP target genes that are regulated by SNIP1 demethylation (SNIP1K301R). d Heatmap representing expression levels of 55 YAP target genes determined by RNA-Seq in (a). Forty genes were found to be significantly downregulated in K301R mutant cell lines were highlighted in red. e qPCR analysis of a group of YAP target genes that were induced upon SNIP1 K301 methylation (SNIP1K301M) (n = 3). f, g these representative typical pathway genes that may be regulated by SNIP1 methylation were measured and verified in MDA-MB-231 (f) and BT549 (g) cells by qRT-PCR (n = 3). h Western blot was performed in MDA-MB-231/sgSNIP1 and BT549/sgSNIP1 cells transfected with SNIP1 sgRNA-resistant Flag-SNIP1WT and SNIP1K301R mutant (n = 3). Data information: In (d, e–g), statistical analysis was performed by a two-tailed t-test. mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001. Panels (e–h) show 1 experiment representative of three independent experiments with similar results.

Fig 4: SNIP1 methylation promotes its oncogenic functions.a A schematic workflow of IAP LC-MS/MS experiments. BT549 and MDA-MB-231 cell lysates were digested to perform IAP LC–MS/MS assays. b MS analysis showing potential methylation sites in SNIP1 after the immunoprecipitation (IP) of SNIP1 in BT549 and MDA-MB-231 cells. c Schematic representation of SNIP1 showing the c-MYC binding domain (FHA) and its amino acid sequence with all lysine (K) residues highlighted in red. NLS, nuclear localization signal. FHA, Forkhead associated domain. d Immunoblot (IB) analysis of the lysine methylation of wildtype (WT) and KR mutant SNIP1 in MDA-MB-231 and BT549 cells transfected with the indicated plasmids; lysates were assessed by IP with anti-HA and IB with anti-Pan-Me-Lys and anti-HA (n = 3). e LC-MS/MS spectrum of the tryptic peptide IADIPIDHPSCSK identified a monomethylated residue at K301, carrying a mass of +14.016 Da. f, g Cells derived from MDA-MB-231/sgSNIP1 cells re-overexpressed with indicated SNIP1-WT or SNIP1-K301R and selected with hygromycin (200 µg/ml) for 72 h before the collection was subjected to mouse xenograft assays by orthotopic injection. Tumor sizes were monitored and analyzed (f, g) (n = 6 mice per group). h Representative photos of Ki-67 positive staining cells in murine primary tumor sections of indicated tumor tissues. Scale bar, 100 µm (IHC). i Quantification of the number of cells positive for Ki-67 staining per field of vision in tumor sections from (h) (n = 6 for each group). FOV field of view. j–m Cells generated in (f) were subjected to mouse xenograft through tail vein injection. Lung metastases were monitored (j, l) and calculated (k, m) (n = 8 mice per group). n Kaplan–Meier survival of mice in (j) (n = 8 mice per group). Data information: In (g, i, k, m), statistical analysis was performed by a two-tailed t-test. In (n), by log-rank test. ***P < 0.001. Data are represented as mean ± SEM. Panels (d) and (f–n) show one experiment representative of three independent experiments with similar results.

Fig 5: KMT5A-mediated SNIP1 K301 methylation promotes TNBC metastasis via activating YAP signaling.a Western blotting for ectopic expression of SNIP1WT or SNIP1K301M mutant combined with KMT5A knockout on MARK4 protein expression and Hippo signaling activation in MDA-MB-231 and BT549 cells. Empty vector (EV), Wild-type (WT) (n = 3). b, c Effects of ectopic expression of SNIP1WT or SNIP1K301M mutant combined with KMT5A knockout on MARK4 mRNA expression (b) and luciferase activity from MARK4 promoter fragments (c). Nontargeting sgRNA (sgC) (n = 3). d Effects of ectopic expression of SNIP1WT or SNIP1K301M mutant combined with KMT5A knockout on MDA-MB-231 and BT549 cells proliferation (n = 3). e Cells generated in (d) were subjected to mouse xenograft assays by orthotopic injection in athymic nude mice, and tumor sizes were monitored and analyzed. Data represent the mean ± SEM (n = 5 mice per group). f–h Effects of ectopic expression of SNIP1WT or SNIP1K301M mutant combined with KMT5A knockout on cell invasion (f) (n = 3), lung metastasis (g), and mouse lifespan (h). 1 × 105 MDA-MB-231 breast cancer cells transduced with control sgRNA (sgC) or sgKMT5A, with or without ectopic expression of SNIP1WT or SNIP1K301M mutant were implanted into the lateral tail vein of athymic nude mice (n = 5 mice per group). Mice were euthanized 25 days after the injection. The number of macroscopic lesions on the lung surfaces were quantified at necropsy from cohorts of mice injected with MDA-MB-231/sgC+EV, MDA-MB-231/sgKMT5A+EV, MDA-MB-231/sgKMT5A with SNIP1WT or SNIP1K301M re-expression breast cancer cells. Independent experimental groups under the same conditions were performed to calculate the animal survival (n = 5 mice per group). Data information: In (b–g), statistical analysis was performed by a two-tailed t-test. In (h), by log-rank test. ***P < 0.001. Data are represented as mean ± SEM. Panels (a–h) show one experiment representative of three independent experiments with similar results.