NONO directly interacts with nuclear PKM2 in TNBC cells
The paraspeckle protein NONO plays a critical role in TNBC, although its direct downstream transcriptional target genes are unknown [13–15]. To identify potential proteins that interact with NONO, we used a proximity-dependent biotinylation identification approach known as BioID2 [25], in which NONO is fused to the N-terminus of the BioID2 vector and transduced into human MDA-MB-231 cells. An empty vector containing only BirA biotin ligase was used as the control. After biotin-streptavidin affinity purification, proteins associated with NONO were analyzed using quantitative mass spectrometry. Among the proteins that were specifically enriched in NONO-BioID2-expressing cells, PKM2 was identified as an NONO-interacting protein in the top 10 list (Fig. 1A, Additional file 2: Table S1). In addition, we found that SFPQ, PSPC1, and MSN [12–14], which are NONO-binding proteins, are also present. To confirm the interaction between NONO and PKM2, we performed co-immunoprecipitation (co-IP) experiments and found that NONO interacted with PKM2 in both MDA-MB-231 and BT-549 cells (Fig. 1B and C). Furthermore, immunofluorescence staining revealed that NONO co-localized with PKM2 in the nuclei of MDA-MB-231 cells (Fig. 1D). These results agree with previous observations that PKM2 is present in the nucleus [20, 21, 30].
Next, we found that NONO directly interacted with PKM2 in the GST pull-down experiments (Fig. 1E). To further characterize the determinants mediating the association between NONO and PKM2, we mapped NONO protein domains using GST pull-down experiments and demonstrated that the NOPS and coiled-coil domains of NONO were sufficient for their interaction (Fig. 1F). Similarly, we demonstrated that the A and C domains of PKM2 were responsible for its interaction with NONO (Fig. 1G), indicating that NONO interacts with PKM2 through multiple domains. Taken together, these data indicate that PKM2 is a novel protein that interacts with NONO in the TNBC cell nuclei.
NONO expression is associated with the prognosis of TNBC patients, and NONO is required for TNBC cell metastasis
To investigate the clinical significance of NONO expression in patients with TNBC, we first analyzed NONO expression by immunohistochemistry (IHC) using tissue microarrays containing 60 TNBC samples and matched adjacent normal tissues. We observed that NONO expression was significantly upregulated in TNBC tissues compared to that in the adjacent normal tissues (Fig. 2A and B). Notably, NONO expression correlated with tumor size and higher-grade lymph node status in patients with TNBC (Fig. 2C; Additional file 2: Table S2). Importantly, Kaplan-Meier survival analysis revealed that TNBC patients with high NONO expression had a shorter overall survival (Fig. 2D). The expression levels of NONO in TNBC tissues were significantly higher than those in non-TNBC tissues (Additional file 1: Fig. S1A). These results indicate that NONO is upregulated in human TNBC tissues and correlates with poor prognosis in patients with TNBC, suggesting that NONO may promote cancer cell invasion during malignant progression.
To determine the effect of NONO on cell growth and invasion, two independent short hairpin RNAs (shRNAs) targeting NONO were used to silence NONO expression. The knockdown efficiency of NONO in human MDA-MB-231 and BT-549 TNBC cell lines was determined by western blot analysis (Fig. 2E). CCK-8 and colony formation assays indicated that the proliferation of MDA-MB-231 and BT-549 cells was significantly inhibited upon NONO knockdown compared with that of scrambled control (Scr) cells (Fig. 2F and G). Transwell assays revealed that the migratory and invasive abilities of TNBC cells were significantly lower in the NONO knockdown group than in the Scr group (Fig. 2H). In addition, the well-established MMTV-PyMT (FVB background) transgenic murine model of spontaneous mammary tumors [31] was used to determine whether NONO directly regulated tumorigenesis. Remarkably, adeno-associated virus (AAV)-mediated NONO depletion in mouse breast tissue was sufficient to attenuate tumor growth (Fig. 2I, J, and K). More importantly, NONO depletion substantially reduced the number of metastatic lung nodules (Fig. 2L and M). These results indicated that NONO is crucial for TNBC growth and metastasis.
PKM2 expression is upregulated in TNBC, and knockdown of PKM2 inhibits TNBC cell metastasis
Similarly, we investigated the clinical significance of PKM2 expression in TNBC patients and found that PKM2 expression was upregulated in human TNBC tissues and correlated with poor prognosis in TNBC patients (Fig. 3A, B, and C; Additional file 1: Fig. S1B and Table S3), which is consistent with previous results [30] and further suggests that PKM2 may promote cancer cell invasion during malignant progression. Furthermore, our in vitro knockdown experiments (Fig. 3D, E, F, and G) and in vivo animal study results (Fig. 3H, I, J, K, and L) indicated that PKM2 is crucial for TNBC growth and metastasis.
SERPINE1 is a key transcriptional target of NONO and PKM2 in TNBC cells
Next, we sought to understand how NONO and PKM2 regulate the migration and invasion of TNBC cells. To identify potential transcriptional targets of the NONO/PKM2 complex, we performed RNA-seq analysis to profile co-regulated target genes. Through integrated analysis of RNA-seq results following NONO and PKM2 knockdown in MDA-MB-231 cells, we found that nearly half of the differentially expressed genes DEGs (451/999) following NONO knockdown were also regulated by PKM2 (Fig. 4A, GSE266177). Further analysis revealed that SERPINE1 (encoding the PAI-1 protein) [22, 23, 32] was the gene whose expression was most downregulated by either NONO or PKM2 knockdown (Fig. 4B, GSE266177). In the top 10 heatmaps, IL11, CCN2, RCN1, VDAC1, CDKN1A (P21), ADAMTSL4, MATN2, FERMT2, and MCAM, which play key roles in cell growth, adhesion, migration, and differentiation [33–40], were significantly downregulated when either NONO or PKM2 was knocked down. Given that PAI-1 facilitates tumor cell detachment from the matrix and promotes tumor dissemination and metastasis, which has been recommended as a promising biomarker for poor prognosis in primary breast cancer patients by the American Society of Clinical Oncology (ASCO) and the European Organization for Research and Treatment of Cancer (EORTC) clinical operation guidelines [22, 23], we selected SERPINE1 as a primary target for further study. We confirmed that NONO knockdown in MDA-MB-231 cells significantly reduced the expression of SERPINE1 at both the transcriptional and protein level (Fig. 4C and D). Subsequently, we examined whether the enforced expression of PAI-1 would compensate for NONO knockdown. We first overexpressed exogenous PAI-1 in NONO-depleted MAD-MB-231 cells (Fig. 4E). We then performed CCK-8, colony formation, and Transwell assays using these cells. We found that the overexpression of PAI-1 significantly rescued the reduced proliferation, migration, and invasion of NONO-depleted cells (Fig. 4F, G, and H). To further support the in vitro results, we performed in vivo analysis using xenograft models in nude mice. We showed that overexpression of PAI-1 significantly reversed the growth defects in NONO-depleted MDA-MB-231 xenograft tumors (Fig. 4I, J, and K).
Similarly, we found that knockdown of PKM2 significantly reduced SERPINE1 transcription in MDA-MB-231 cells (Fig. 4L and M). The proliferative, migratory, and invasive capabilities of PKM2-depleted MDA-MB-231 cells could be rescued by ectopic expression of PAI-1 (Fig. 4N, O, P, and Q). Consistent experimental results were obtained regarding the roles of NONO and PKM2 in human BT-549 TNBC cells (Additional file 1: Fig. S2A-L).
To further investigate the clinical relevance of PAI-1 expression in patients with TNBC, we examined its expression using IHC on tissue microarrays containing 60 pairs of TNBC samples and their matched adjacent normal tissues. We demonstrated that PAI-1 was notably upregulated in TNBC tissues compared to adjacent normal tissues (Additional file 1: Fig. S2M and N). Importantly, the expression levels of NONO and PAI-1, as well as those of PKM2 and PAI-1, were significantly positively correlated in human TNBC samples according to Pearson correlation analysis (Fig. 4R), further confirming that SERPINE1 is the downstream target gene of NONO/PKM2 in TNBC cells. Collectively, these data suggest that NONO/PKM2 promotes TNBC progression by activating SERPINE1 transcription.
NONO-dependent PKM2 and its nuclear protein kinase activity are critical for SERPINE1 transcription and TNBC progression
Given that NONO and PKM2 positively regulate SERPINE1 transcription in TNBC cells, and that they directly interact in the nucleus, we investigated how they regulate SERPINE1 expression. As expected, enforced the overexpression of PKM2 enhanced SERPINE1 expression. However, this increase in expression was abolished by NONO knockdown (Fig. 5A and B). Next, we examined whether PKM2-mediated H3T11ph modifications at the SERPINE1 promoter were enriched in a NONO-dependent manner. ChIP‒qPCR revealed a significant reduction in H3T11ph enrichment at the SERPINE1 promoter in the NONO knockdown group compared to that in the negative control (NC) group (Fig. 5C). Previous studies have demonstrated that PKM2 exhibits pyruvate kinase or protein kinase activity, which depends on its cytosolic tetramer or nuclear dimer state [19, 41, 42]. To further explore whether the protein kinase of PKM2 is crucial for SERPINE1 transcriptional activation, we utilized two well-established PKM2 mutants, PKM2-Y105F (predominant tetramer form harboring pyruvate kinase activity) [42] and PKM2-R399E (predominant dimer form harboring protein kinase activity) [19] to examine their effects on SERPINE1 expression. We overexpressed PKM2-WT, PKM2-Y105F, or PKM2-R399E in PKM2-depleted MDA-MB-231 cells. We found that the PKM2-Y105F mutant did not activate SERPINE1 expression, whereas the PKM2-R399E mutant had an activating effect on SERPINE1 expression similar to that of PKM2-WT (Fig. 6D and E). These results suggest that the protein kinase activity of PKM2 is key to activating SERPINE1 expression in the nucleus.
To identify the potential key residues of PKM2 responsible for NONO interactions, we used the ZDOCK server (https://zdock.umassmed.edu/) for molecular docking analyses. Molecular docking confirmed good and stable binding of NONO to PKM2 (Additional file 1: Fig. S3A) and revealed seven positions of PKM2 that may play important roles in the interaction between PKM2 and NONO (Additional file 1: Fig. S3B). GST pull-down assays demonstrated that the PKM2-S406A mutant (Ser to Ala at aa 406) significantly reduced this interaction, whereas the interactions of PKM2-R400A, T412A, D476A, D487A, W515A, and R526A mutants with NONO were similar to those of wild-type PKM2 (PKM2-WT, Fig. 5F). To explore the effect of the S406A mutation of PKM2 on TNBC progression, we overexpressed PKM2-WT or the PKM2-S406A mutant in MDA-MB-231 cells. Consistently, the PKM2-S406A mutant immunoprecipitated significantly less NONO in the MDA-MB-231 cells (Fig. 5G). Notably, the PKM2-S406A mutant exhibited the same subcellular distribution as that of PKM2-WT (Fig. 5H). However, compared with PKM2-WT, PKM2-S406A overexpression in MDA-MB-231 cells failed to activate SERPINE1 expression (Fig. 5I and J). In addition, we found that compared to PKM2-WT, PKM2-S406A overexpression in MDA-MB-231 cells significantly inhibited cell growth, migration, and invasion (Fig. 5K, L, and M). Taken together, these results demonstrated that NONO-dependent PKM2 and its nuclear protein kinase activity are crucial for SERPINE1 expression and TNBC cell invasion.
PKM2-mediated H3T11ph cooperates with TIP60-mediated H3K27ac to promote SERPINE1 expression
A previous study has revealed that PKM2 mediates H3T11ph, which is involved in transcriptional regulation [21]. To explore the effect of PKM2 on other histone modifications, we examined global changes in other key histone H3 modifications upon PKM2 knockdown in MDA-MB-231 cells. As expected, the levels of H3T11ph were markedly decreased upon PKM2 knockdown (Fig. 6A). Interestingly, we found that the levels of H3K27ac, a promoter and enhancer marker [43–45], were markedly lower in PKM2-knockdown cells than in NC cells (Fig. 6A). Additionally, we observed that the levels of the histone markers H3K4me1 and H3K9ac, which have been previously described [21], decreased upon PKM2 knockdown (Fig. 6A). There were no obvious changes in the levels of H3T3ph, H3K4me2/3, H3K4ac, H3K9me3, H3S10ph, H3K14ac, H3K14la, H3R17me2s, H3K18ac, or H3K27me2/3 modifications between PKM2 knockdown cells and NC cells (Fig. 6A). These data suggested that PKM2-mediated H3T11ph cooperates with H3K27ac to regulate gene transcription in TNBC cells.
To understand how NONO, H3T11ph, and H3K27ac collaborate to regulate gene transcription, we performed cleavage under targets and tagmentation (CUT&Tag) experiments in MDA-MB-231 cells to determine the enrichment profiles of NONO, H3T11ph, and H3K27ac in the whole genome. Interestingly, we found that NONO, H3T11ph, and H3K27ac signals were significantly enriched at transcription start sites (TSSs), implying that they may perform transcriptional regulatory functions (Fig. 6B, GSE266179). In addition, H3T11ph and H3K27ac signals were markedly reduced in PKM2-KD cells compared to their respective NC cells (Fig. 6B), which was in good agreement with our western blot analysis results (Fig. 6A). In fact, we found that NONO, H3T11ph, and H3K27ac were co-enriched in the promoter, TSS, and 3’-end regions of SERPINE1 (Fig. 6C). These results were further confirmed by ChIP‒qPCR in MDA-MB-231 cells (Fig. 6D). Notably, NONO enrichment largely overlapped with H3T11ph peaks genome-wide in MDA-MB-231 cells (Additional file 1: Fig. S4A, GSE266179). Moreover, through bioinformatics analysis, we found that the genome-wide occupancy of NONO and H3T11ph, H3T11ph, and H3K27ac was highly correlated, although there was no significant correlation between NONO and H3K27ac (Additional file 1: Fig. S4B). These results further suggest that PKM2-mediated H3T11ph could be enriched in promoters through NONO recruitment of PKM2, where H3T11ph cooperates with H3K27ac to activate SERPINE1 transcription.
Next, we explored the possible link between H3T11ph and H3K27ac. We searched the RNA-seq data obtained upon PKM2 knockdown (GSE266177) and examined changes in the expression levels of histone acetyltransferases KAT5 (encoding TIP60), KAT3B (encoding PCAF), CREBBP (encoding CBP), and EP300 (encoding P300), which are associated with histone acetylation. We then performed western blot analyses and found that PKM2 knockdown significantly reduced the expression of TIP60 but upregulated P300 expression in MDA-MB-231 cells, whereas the expression of PCAF and CBP remained unchanged compared with that in NC controls (Additional file 1: Fig. S5A). Therefore, we selected KAT5/TIP60 for further study. RT-qPCR assays confirmed that KAT5 mRNA levels significantly decreased following PKM2 knockdown (Additional file 1: Fig. S5B). Similar results were obtained for the BT-549 cells (Additional file 1: Fig. S6A and B). In addition, our CUT&Tag data demonstrated that H3T11ph modifications were enriched in the KAT5 promoter and that H3T11ph enrichment was significantly reduced in the promoter upon PKM2 knockdown (Additional file 1: Fig. S5C). Consistent results were obtained by ChIP‒qPCR in MDA-MB-231 cells (Additional file 1: Fig. S5D). These data indicated that PKM2 directly activates KAT5 transcription.
To determine whether TIP60 regulates SERPINE1 expression, we knocked down KAT5 expression in MDA-MB-231 cells. KAT5 knockdown significantly reduced SERPINE1 expression in MDA-MB-231 cells compared to that in NC control cells (Additional file 1: Fig. S5E and F). As expected, the levels of H3K27ac were markedly reduced following KAT5 knockdown (Additional file 1: Fig. S5F). Importantly, the levels of PAI-1 and H3K27ac in PKM2-depleted MDA-MB-231 cells were partially restored by ectopic TIP60 expression (Additional file 1: Fig. S5G). These results were also observed in BT-549 cells (Additional file 1: Fig. S6C, D and E). In addition, we analyzed the expression profiles extracted from GEO datasets (GSE76275) and found that the expression of PKM2 and KAT5 was positively correlated in human TNBC samples (Additional file 1: Fig. S6F). Taken together, these results suggest that PKM2 can also transcriptionally regulate KAT5 expression and that TIP60-mediated H3K27ac cooperates with PKM2-mediated H3T11ph to activate SERPINE1 expression.
NONO or PKM2 deficiency reduces PAI-1 expression and inhibits the malignant progression of spontaneous mammary tumors in mice
The nuclear protein NONO is highly conserved in humans and mice, and shares 98% amino acid sequence similarity [7, 8]. To further assess the role of NONO in regulating mammary tumor progression in vivo, we constructed a spontaneous mammary tumor mouse line in which NONO was specifically knocked out in the breast tissue (Fig. 7A). Consistent with our previous results, conditional loss of NONO in mammary tissue significantly inhibited tumor growth (Fig. 7B and C; Additional file 1: Fig. S7A). In addition, knockout of NONO markedly reduced the lung metastatic capacity and suppressed the formation of larger metastatic nodules in spontaneous mammary tumor model mice (Fig. 7D). The intratumoral levels of NONO, PAI-1, and Ki67 were analyzed using western blotting and immunohistochemical (IHC) staining. Conditional mammary NONO depletion significantly reduced PAI-1 and Ki67 expression (Fig. 7E and F; Additional file 1: Fig. S7B).
We also generated a mouse model of spontaneous breast cancer with mammary-specific PKM2 knockout (Fig. 7G). Similarly, tumor growth and lung metastatic capacity were significantly inhibited by conditional PKM2 knockout in breast tissue (Fig. 7H, I, and J; Additional file 1: Fig. S7C). The intratumoral levels of PKM2, PAI-1, and Ki67 were analyzed using western blotting and immunohistochemical (IHC) staining. Conditional mammary PKM2 depletion significantly reduced PAI-1 and Ki67 expression (Fig. 7K and L; Additional file 1: Fig. S7D). Taken together, these in vivo data reinforce the notion that NONO and PKM2 are critical for the malignant progression of breast tumors via SERPINE1 transcriptional activation.