Blocking ubiquitination of hnRNPA1 maintains the self-renewal of breast cancer stem cells via mutually exclusive splicing of PKM pre-mRNA

Blocking ubiquitination of hnRNPA1 maintains the self-renewal of breast cancer stem cells via mutually exclusive splicing of PKM pre-mRNA | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Blocking ubiquitination of hnRNPA1 maintains the self-renewal of breast cancer stem cells via mutually exclusive splicing of PKM pre-mRNA Miao He, Xuemei Lv, Han Li, Wei-Wei Tong, Yuanyuan Yan, Xiaoyu Sun, and 10 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7722676/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 14 Apr, 2026 Read the published version in Oncogene → Version 1 posted 11 You are reading this latest preprint version Abstract PKM serves as a rate-limiting enzyme in glycolysis, which produces two isoforms depending on the inclusion of either exon 9 (PKM1) or exon 10 (PKM2). The M2 pyruvate kinase (PKM2) isoform is commonly upregulated in various cancers, where it plays a pivotal role in regulating Warburg effect. Breast cancer stem cells (BCSCs) exhibit enhanced glycolysis, which is crucial for their self-renewal. However, the specific role of PKM2 in BCSCs remains largely unexplored. Here, we report that PKM2 expression is upregulated in BCSCs. Meanwhile, we identify that LINC00887 is significantly upregulated in BRCA through a genome-wide LNCRNA microarray. Moreover, we recognize that hnRNPA1 interacts with PKM pre-mRNA and regulates its mutually exclusive splicing. Furthermore, we demonstrate that LINC00887 maintains the self-renewal of BCSCs by promoting PKM2 splicing and reprogramming glucose metabolism. Mechanistically, LINC00887 upregulates PKM2 expression by binding hnRNPA1, thereby concealing its ubiquitination site, which blocks its ubiquitination and maintains its stability. Consistently, overexpression of hnRNPA1 almost completely rescues/reverses the inhibitory effects of LINC00887 KD in BRCA.Collectively, our study characterizes the LINC00887/hnRNPA1/PKM1/2 axis in BRCA and reveals the essential role of LINC00887 in BCSCs self-renewal/maintenance through promoting hnRNPA1-mediated PKM2 splicing, highlighting the therapeutic potential of targeting cancer metabolism. Biological sciences/Stem cells/Cancer stem cells Biological sciences/Cancer/Breast cancer Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Breast cancer (BRCA) represents a heterogeneous disease, characterized by diverse subtypes and clinical response 1 , 2 . Within this complexity, research on cellular hierarchies and intratumor heterogeneity provides support for the existence of breast cancer stem cells (BCSCs), characterized by unlimited self-renewal/repopulating potential, which contribute to the initiation and progression of the disease, as well as to treatment failure and recurrence of BRCA 3 – 8 . Targeting and eliminating BCSCs is considered a promising strategy for BRCA treatment. However, current therapeutic strategies inadequately target BCSCs. Therefore, understanding the biology and regulation of BCSCs holds significant implications for developing more effective therapeutic strategies targeting this resilient cell population. Reprogramming of cellular metabolism is widely considered a hallmark of cancer 9 , 10 . In contrast to normal healthy cells, tumor cells predominantly rely on glycolysis even in the presence of oxygen, which is termed as "aerobic glycolysis" or the "Warburg effect" 11 . Emerging evidence highlights that BCSCs exhibit distinct metabolic features, especially enhanced glycolytic activity, which enables them to adapt to nutrient scarcity and contribute to their survival and aggressiveness 12 – 14 . Pyruvate kinase muscle isozymes (PKMs) serve as rate-limiting enzymes in glycolysis, which catalyzes the conversion of phosphoenolpyruvate (PEP) to pyruvate. Pyruvate kinase muscle isozymes M1 (PKM1) and M2 (PKM2), are derived from mutually exclusive splicing of exons 9 and 10 of PKM gene, with PKM1 including exon 9 and PKM2 including exon 10. It is reported that a splice isoform switch from PKM1 to PKM2 is essential for the transition of cellular metabolism towards aerobic glycolysis, thereby fostering tumorigenesis 15 . Alternative splicing, a critical mechanism in gene expression, relies on a complex array of splicing factors. Evidence shows that heterogeneous nuclear ribonucleoprotein (hnRNP) proteins, including hnRNPA1, and hnRNPA2 are involved in promoting PKM2 splicing 16 . Nevertheless, the regulatory mechanisms underlying PKM2 splicing in BCSCs remain elusive. As one of the most prevalent post-translational modifications, ubiquitination fine-tunes the fate and function of substrate proteins by regulating their stability, localization, activity, and interactions with other proteins 17 . Long noncoding RNAs (LNCRNAs) are versatile regulators of gene expression with functions spanning various levels of cellular regulation, from transcriptional and post-transcriptional processes to chromatin organization and cell signaling 18 . Evidence reveals that LNCRNAs play multifaceted roles in regulating protein ubiquitination, as they bind directly to proteins, block ubiquitination sites, modulate enzyme activity, and act as scaffolds or guides for protein complexes involved in ubiquitination 19 – 22 . Dysregulation of LNCRNAs is closely associated with various cancer types, including breast cancer 23 , 24 . Previous studies have revealed that hnRNPs undergo ubiquitination and subsequent proteasomal degradation 25 – 27 . However, whether LNCRNAs regulate the ubiquitination of hnRNPs has yet to be addressed. In this work, we performed a genome-wide expression profiling in BCSCs and identified LINC00887 as a key regulator of BRCA stemness. We showed that the expression of hnRNPA1 was markedly upregulated in BCSCs, where it binds to the flanking sequence of PKM pre-mRNA exon 9, inducing a switch to the PKM2 isoform, a crucial requirement for the self-renewal of BCSCs. Furthermore, we identified that LINC00887 prevents ubiquitination-mediated hnRNPA1 degradation to enhance aerobic glycolysis by regulating mutually exclusive splicing of PKM pre-mRNA. These data highlight targeting cancer metabolism as a promising therapeutic strategy for BRCA. Results The high expression of LINC00887 is positively correlated with PKM2-mediated glycolysis in BCSCs To explore the pathogenesis of BCSCs, we utilized serum-free suspension culture to enrich the subpopulation of CD44 + /CD24 − MCF-7 mammospheres (MCF-7 MS) with BCSC-like properties, then performed microarray on MCF-7 MS along with its parental MCF-7 cells (Fig. 1 A). We identified thousands of dysregulated genes in MCF-7 MS (Supplementary Fig. S1 A). Notably, gene set enrichment analyses (GSEA) revealed significant enrichment for glycolysis in MCF-7 MS (Fig. 1 B). To further corroborate this discovery, we detected glycolytic rates by XF glycolytic stress assays, as well as determining lactate levels in MS and its parental cells. Consistently, MS showed elevated glycolytic rates and higher lactate levels compared to its parental cells (Fig. 1 C- 1 E). These findings strongly suggest that BCSCs exhibit active aerobic glycolysis, heavily relying on glycolytic metabolism to sustain their energy requirements and other vital biological functions. Prior investigations into the transcriptomic diversity of BCSCs have primarily focused on mRNA, little is known about LNCRNA heterogeneity. To identify the LNCRNAs involved in the maintenance of BCSCs properties, we performed genome-wide LNCRNA microarray on MCF-7 MS along with its parental MCF-7 cells (Fig. 1 A). Principal component analysis (PCA) and cluster analysis showed that the MCF-7 MS group could be separated from the parental MCF-7 cells group, indicating a distinct LNCRNA signature (Supplementary Fig. S1 B). Further analysis of microarray data identified hundreds of differentially expressed LNCRNAs, including 759 down-regulated and 632 up-regulated LNCRNAs in MCF-7 MS compared to parental MCF-7 cells (Supplementary Fig. S1 C). We focused on the top 10 MCF-7 MS-enriched LNCRNAs to determine prognostic LNCRNAs in breast cancer (Fig. 1 F), found that both the expression of LINC00887 and MGC27382 significantly correlate with poorer survival in breast cancer patients (n = 1538, http://kmplot.com/analysis/ ) (Fig. 1 G and Supplementary Fig. S1 D). Additionally, RT-qPCR validation showed a more than 2-fold upregulation in LINC00887 expression within mammospheres compared to its parental cells, while MGC27382 exhibited lower expression levels compared to LINC00887 (Fig. 1 H and Supplementary Fig. S1 E). In our in-house tissue microarray, LINC00887 is aberrantly overexpressed in BRCA tissues relative to tumor-adjacent control (n = 136, Fig. 1 I). Further analyses revealed that elevated LINC00887 level was positively correlated with tumor size and advanced AJCC stage (Supplementary Fig. S1 F and S1G). Strikingly, LINC00887 expression was significantly elevated in CD44⁺/CD24⁻ BCSCs (Fig. 1 I and 1 J), suggesting that LINC00887 may contribute to the maintenance of BCSC properties. Consistently, our in vitro functional studies revealed that LINC00887 KD dramatically inhibited cell proliferation, reduced the capacity of cellular sphere formation and the proportion of CD44 + CD24 − BCSCs, whereas ectopic expression of LINC00887 markedly increased anchorage-dependent growth and anchorage-independent growth capacities (Supplementary Fig. S2 A-S2I). As expected, in vivo limited dilution assay confirmed that LINC00887 KD led to dramatic decreases in both tumorigenic ability and the frequency of BCSCs (Fig. 1 K- 1 N and Supplementary Fig. S2 J-S2K). Moreover, to identify biological pathways associated with LINC00887, we conducted GSEA enrichment analysis using TCGA BRCA data (n = 1109). Notably, glycolysis signaling pathways are hyper-activated in patients with high LINC00887 expression (Fig. 1 O), indicating that LINC00887 may support the maintenance of BCSC properties by modulating glycolysis pathway. To further determine critical metabolic target genes of LINC00887, we conducted screening on glycolytic enzymes (Supplementary Fig. S2 L). Among all the genes examined, PKM1 and PKM2 were found to be the most significantly changed by LINC00887 KD (Fig. 1 P). PKM1 and PKM2 are two alternative splicing isoforms of pyruvate kinase muscle (PKM), serving as rate-limiting enzyme in glycolysis. In various cancer cells, PKM2 predominates as the primary form of PKM to modulate cancer metabolism 15 , 28 , 29 . We further investigated the clinical relevance of PKM isoforms and found that, compared to normal control, PKM2 expression was significantly elevated in BRCA, whereas PKM1 expression was markedly reduced (Fig. 1 Q). Additionally, high PKM2 expression was positively correlated with advanced tumor Stage, while low PKM1 expression correlated with higher tumor Stage (Supplementary Fig. S2 M and S2N). We also found that PKM2 protein levels were higher in the BRCA CD44 + CD24 − subsets, while PKM1 expression remained low (Fig. 1 Q). Further correlation analysis confirmed a remarkably positive relationship between LINC00887 expression and PKM2 expression, while PKM1 showed a mild negative correlation with LINC00887 (Fig. 1 R), implying that PKM2 and PKM1 are the potential targets of LINC00887. Together, these findings indicate that LINC00887 might activate the glycolytic pathway of BCSCs by regulating PKM1 and PKM2. LINC00887 promotes BCSC properties by activating PKM2-mediated glycolysis The PKM gene encodes two major isoforms: PKM1 and PKM2. Significant elevation of PKM2 expression was observed in various cancers, whereas the tissue-specific enzyme expressions of PKM1 was mainly expressed in normal cells. It is thought that PKM2 rather than PKM1 promotes glycolysis and tumor progression in cancer. However, the mechanisms driving the shift in PKM isoform expression, particularly in BCSCs, remain poorly understood. Here, we observed a marked increase in PKM2 protein levels and a corresponding decrease in PKM1 in BRCA compared with matched adjacent normal tissues (Fig. 1 Q). Moreover, we revealed that PKM2, but not PKM1, was highly expressed in CD44 + CD24 − BRCA (Fig. 1 Q). Consistently, Western blot further confirmed the upregulation of PKM2 and the downregulation of PKM1 in BCSCs (Fig. 2 A and 2 B), highlighting a potential role for PKM2 in the metabolic reprogramming of BCSCs. To investigate the regulatory mechanism underlying PKM isoform expression, we focused on the long noncoding RNA. LINC00887 KD significantly increased PKM1 protein levels and decreased PKM2 levels in MCF-MS cells, whereas forced expression of LINC00887 in MCF-7 cells had the opposite effect (Fig. 2 C). Functionally, LINC00887 KD resulted in a significant reduction in glycolytic rates and lactate levels, while forced expression of LINC00887 significantly increased glycolytic rates and lactate levels (Fig. 2 D- 2 G). Collectively, our data suggest that LINC00887 promotes glycolysis in BCSCs by regulating PKM isoform expression. PKM two isoforms arise from mutually exclusive alternative splicing of the PKM pre-mRNA, which reflects the inclusion of either exon 9 (PKM1) or exon 10 (PKM2) (Fig. 2 H). We analyzed the distribution of these two isoforms of PKM and found that that PKM2, rather than PKM1, predominates in BCSCs (Fig. 2 I). To determine whether LINC00887 mediates PKM1/PKM2 splicing, we detected the altered splicing of PKM by semi-quantitative RT-PCR followed by exon10-specific restriction digestion. As shown in Fig. 2 J, LINC00887 KD notably augmented the splicing of the PKM1 variant while diminishing the PKM2 isoform. Conversely, there was a substantial decrease in PKM1 splicing and an increase in PKM2 splicing in MCF7 cells overexpressing LINC00887 (Fig. 2 J). Taken together, our results indicate that PKM1 and PKM2 are functionally essential metabolic targets of LINC00887 and contribute to LINC00887 KD-induced glycolytic inhibition in BCSCs. HnRNPA1 drives PKM alternative splicing to favor PKM2 isoform expression in BCSCs Alternative splicing regulation involves recognition of cis-acting sequence motifs within pre-mRNA by trans-acting RNA binding proteins (RBPs) 30 . PKM1 and PKM2 splice variants result from the recruitment of specific splicing factors to PKM pre-mRNA, thereby driving mutually exclusive alternative splicing events. To identify potential splicing factors involved in the alternative splicing of PKM pre-mRNA in BCSCs, we compared the expression of splicing factors in MCF-7 MS vs MCF-7 cells (Fig. 3 A). hnRNPA1 and QKI were two of the most highly expressed splicing factors in MCF-7 MS cells relative to MCF-7 cells (Fig. 3 B and Supplementary Fig. S3A). MOE docking analysis revealed that both hnRNPA1 and QKI bind to PKM pre-mRNA, with hnRNPA1 exhibiting a substantially higher binding affinity (S = − 63.06) than QKI (S = − 44.07) (Fig. 3 C- 3 D and Supplementary Fig. S3B). Further structural analysis revealed that hnRNPA1 recognizes the UAGGG motif located within the flanking region of exon 9 (Fig. 3 D and 3 E). Subsequent RNA pull-down assays using biotin-labeled PKM probes confirmed the direct interaction of wild-type (WT) PKM, but not mutant (MUT) PKM, with the hnRNPA1 protein (Fig. 3 F). Furthermore, forced expression of hnRNPA1 promotes trans-expression of PKM1 to PKM2 (Fig. 3 G). Similar patterns were observed by western blotting in hnRNPA1 overexpressing MCF7 cells (Fig. 3 H and 3 I). To evaluate the clinical relevance of the hnRNPA1/PKM2 axis, we checked the correlation between hnRNPA1 and PKM2 expression in BRCA patient cohorts. HnRNPA1 expression showed a positive correlation with PKM2 expression (Fig. 3 J), supporting the regulation of PKM2 by hnRNPA1. GSEA analysis of TCGA BRCA data revealed that glycolysis-related gene signatures were significantly enriched in hnRNPA1⁺ samples (Fig. 3 K). As expected, forced expression of hnRNPA1 significantly enhanced glycolytic rates (Fig. 3 L). Furthermore, we investigated the clinical relevance of hnRNPA1 protein and found that compared to normal control, hnRNPA1 protein was highly expressed in BRCA (Supplementary Fig. S3C-S3E). Moreover, hnRNPA1 expression was elevated in CD44 + CD24 − BRCA than in non-CD44 + CD24 − BRCA (Fig. 3 M). Consistently, western blot assays revealed that the expression of hnRNPA1 was markedly upregulated in BCSCs (Fig. 3 N). KM Plotter analysis showed that high hnRNPA1 protein expression was associated with poor overall survival in BRCA patients (n = 108, P < 0.05) (Supplementary Fig. S3F). Further analysis revealed that elevated hnRNPA1 expression was positively associated with adverse clinical features, including advanced stage (Stage III, P 2.5cm ( P < 0.05), and lymphatic metastasis ( P < 0.05) (Fig. 3 O and Supplementary Fig. S3G-S3I). Collectively, these findings demonstrate that hnRNPA1 directly binds PKM pre-mRNA and promotes mutually exclusive splicing favoring PKM2 production, thereby enhancing glycolysis in BRCA. LINC00887 directly binds to hnRNPA1 to mediate its expression and sustain BSCSs properties Given that both LINC00887 and hnRNPA1 are involved in mutually exclusive splicing of PKM pre-mRNA, we investigated whether LINC00887 promotes PKM2 isoform expression via hnRNPA1. Linear regression analysis revealed a significant positive correlation between LINC00887 and hnRNPA1 protein expression in BRCA (n = 136, r 2 = 0.34, P < 0.0001) (Fig. 4 A). To further elucidate the underlying interaction of hnRNPA1 in the context of LINC00887 mediated BCSCs properties, we employed tartaglialab ( http://www.tartaglialab.com/ ) to evaluate the binding propensity between LINC00887 nucleotide sequences and hnRNPA1 amino acid residues. LINC00887 (2000 nt-2500 nt) exhibited strong interaction signal with hnRNPA1 (Fig. 4 B). MOE docking analysis predicted that LINC00887 could bind to the lysine residues 144–145 in the RRM domain of hnRNPA1(S=-66) (Fig. 4 C). Additionally, we conducted RIP assays and confirmed that hnRNPA1 protein strongly binded with LINC00887 (Fig. 4 D). Interestingly, LINC00887 KD did not alter the mRNA level of hnRNPA1 (Fig. 4 E) but significantly reduced its protein levels (Fig. 4 F), suggesting that LINC00887 may regulate hnRNPA1 at the post-translational level. Conversely, hnRNPA1 overexpression did not alter LINC00887 expression (Fig. 4 G). Collectively, our data suggest that hnRNPA1 is a downstream target of LINC00887. To determine the roles of hnRNPA1 in LINC00887-mediated cell proliferation and BCSCs self-renewal, we ectopically expressed hnRNPA1 in LINC00887 KD MS (Fig. 4 G and 4 H) to conduct rescue assays. As expected, forced expression of hnRNPA1 largely rescued the inhibitory effects of LINC00887 KD on cell proliferation/growth in MCF7 MS and T47D MS (Fig. 4 I). Moreover, forced expression of hnRNPA1 also significantly rescued LINC00887 KD-induced inhibitory effect on the biological characteristics of BCSCs, including the percentage of CD44 + CD24 − populations, and the self-renewal abilities in both MCF-7 MS and T47D MS cells (Fig. 4 J and 4 K). Taken together, our data indicate that hnRNPA1 are bona fide and functionally essential targets of LINC00887 and are responsible for LINC00887 KD-induced inhibitory effects on self-renewal/repopulation capacity in BCSCs. Inhibition of hnRNPA1 ubiquitination by LINC00887 trigger PKM2 splicing and glycolysis Activation Our data showed that hnRNPA1 is required for LINC00887-driven BCSC maintenance. Next, we sought to elucidate the mechanism by which LINC00887 regulates hnRNPA1 protein levels in BCSCs (Fig. 5 A). Our results showed that LINC00887 KD significantly decreased hnRNPA1 protein expression, without affecting its mRNA levels (Fig. 4 E and 4 F), implying that LINC00887 likely modulates hnRNPA1 through a post-translational mechanism. Accumulating evidence suggests that the protein stability of hnRNPA1 is precisely controlled by ubiquitin-proteasome system (UPS) 25 , 26 , 31 . We further identified potential ubiquitination sites on hnRNPA1, specifically at lysine residues 105, 144, 145, 179, and 183, using the BDM-PUB database ( http://bdmpub.biocuckoo.org/ ) (Fig. 5 B). MOE Docking analysis predicted a binding interaction between LINC00887 and hnRNPA1 protein (Fig. 4 C). RNA pull-down assays further confirmed that the WT 887 probe, but not the Mut 887 probe, significantly pulled down endogenous nuclear hnRNPA1 protein, which enrichment was notably diminished following the knockdown of LINC00887 (Fig. 5 C). Strikingly, LINC00887 binds to the lysine 144–145 site of hnRNPA1 (Fig. 4 C), which corresponds to its predicted ubiquitination sites. Functionally, overexpression of LINC00887 led to a noticeable decrease in the ubiquitination of endogenous hnRNPA1 (Fig. 5 D). Moreover, cycloheximide (CHX) chase assays demonstrated that LINC00887 overexpression significantly prolonged the half-life of hnRNPA1 protein in MCF-7 cells (Fig. 5 E and 5 F), supporting its role in stabilizing hnRNPA1 by suppressing its degradation. Taken together, our data suggest that LINC00887 directly binds with hnRNPA1, inhibits its ubiquitination and in turn stabilizes hnRNPA1 in BRCA. To determine the role of hnRNPA1 in LINC00887 KD-induced glycolytic inhibition in BCSCs, we conducted rescue assays by overexpressing hnRNPA1 in LINC00887-depleted cells. First, we detected the expression of PKM2 and PKM1 after co-transfecting MCF-7 MS and T47D MS cells with LINC00887 silencing and hnRNPA1 overexpression lentivirus. Western blot assays confirmed that there was a significant downregulation of PKM2 expression and an upregulation of PKM1 expression in the sh887 group (Fig. 5 G). Notably, overexpression of hnRNPA1 could reverse these changes in protein expression induced by LINC00887 KD (Fig. 5 G and Supplementary Fig. S4A), indicating that LINC00887 mediates the expression of PKM1 and PKM2 in a hnRNPA1-dependent manner. Moreover, we observed that forced expression of hnRNPA1 could rescue LINC00887 KD-induced reduction of CD44 (Fig. 5 G), demonstrating that hnRNPA1 is essential for the LINC00887-mediated self-renewal of BCSCs. Further analysis revealed that overexpression of hnRNPA1 rescued the reduced lactate accumulation induced by silencing LINC00887 in BCSCs (Fig. 5 H). Strikingly, overexpressing hnRNPA1 also restored the glycolytic capacities impaired by LINC00887 KD in MCF-7 MS (Fig. 5 I and Supplementary Fig. S4B). These results strongly confirmed that the LINC00887/hnRNPA1/PKM2 axis activates glycolysis and contributes to the maintenance of BCSCs stemness. Silencing LINC00887 reduces stemness and the growth of BCSCs xenografts via inhibiting hnRNPA1 splicing to form PKM2 formation Finally, we used cell-line-derived xenograft (CDX) models to evaluate the effect of LINC00887-hnRNPA1-PKM2 axis on BRCA progression in vivo . We stably expressed hnRNPA1 in sh887 and shCtrl MCF-7 MS, injected 3x10^5 cells into 6-week-old BALB/c nude female mice and monitored BRCA progression via bioluminescent imaging (Fig. 6 A). As expected, LINC00887 KD significantly reduced the bioluminescence signals, dramatically inhibited tumor growth, and substantially prolonged the overall survival in the recipient mice (Fig. 6 B- 6 G). Forced expression of hnRNPA1 could totally reverse the LINC00887 KD-induced phenotypes in vivo (Fig. 6 B- 6 G), demonstrating that the inhibitory effect of LINC00887 KD on BRCA progression in vivo could be largely due to the dysregulation of hnRNPA1. In addition, immunohistochemistry revealed that LINC00887 KD markedly downregulated the protein levels of PKM2, while significantly increasing the protein levels of PKM1 (Fig. 6 H and 6 I). Meanwhile, we further confirmed that forced expression of hnRNPA1 could rescue LINC00887 KD-induced reduction of PKM2 and CD44, and the increase of PKM1 by western blot (Fig. 6 J and 6 K). Also, we found that LINC00887 KD showed consistently significant inhibitory effects on glycolytic rates in vivo (Fig. 6 L). More strikingly, glycolytic inhibition caused by LINC00887 KD could be largely rescued by overexpression of hnRNPA1 in vivo (Fig. 6 L). Collectively, our data indicate that hnRNPA1 and PKM2 are functionally essential downstream targets of LINC00887 and are responsible for its oncogenic role in BRCA progression. Discussion It is well established that BCSCs play critical roles in BRCA progression and recurrence. Understanding the molecular determinants of BCSCs will help develop targeted strategies and improve clinical outcomes in BRCA patients. Glycolysis reprogramming is an emerging hallmark of BRCA, contributing to the maintenance of tumorigenic potential. Recent advancements have disclosed this metabolic phenotypic difference between cancer cells and their normal counterparts, offering promising opportunities to target key metabolic enzymes in cancer cells 32 , 33 . Here, we revealed that BCSCs exhibit enhanced glycolysis, which is strongly dependent on PKM2. Moreover, we reported that lncRNA heterogeneity contributes to metabolic reprogramming in BRCA, wherein LINC00887 regulates key metabolic enzymes such as PKM1 and PKM2. Furthermore, we demonstrate that LINC00887 blocks the ubiquitination of hnRNPA1 and subsequently switches PKM1 to PKM2 and enhances aerobic glycolysis, which supports the self-renewal and survival of BCSCs. Through transcriptome-wide microarray and Seahorse Glycolytic Rate Assay, we revealed that BSCSs are characterized by their enhanced glycolytic activity, a feature that distinguishes them from non-stem cancer cells and highlights their metabolic plasticity. Emerging data have suggested that the dysregulation of the expression and activities of key central metabolic enzymes contribute to metabolic reprogramming in cancer. PKMs serve as a crucial rate-limiting step in glycolysis, determining the fate of pyruvate and the flow of carbon through this central metabolic pathway. PKM exists in two isoforms, PKM1 and PKM2, which arise from the alternative splicing of exons 9 and 10 of the PKM gene, respectively 15 . While PKM1 is prevalent in most differentiated tissues, PKM2 is predominantly expressed in proliferating tissues and tumors, where it facilitates glycolytic flux and anabolic growth. Recent works on PKM in cancers revealed that switching PKM2 to PKM1 reverses aerobic glycolysis to oxidative phosphorylation and delays tumorigenesis, suggesting PKM2 as an appealing target for cancer therapy 15 . Here, we revealed that PKM2 is significantly upregulated in BRCA particularly in CD44⁺CD24 − BCSCs, whereas PKM1 expression remains low. Importantly, KD of either LINC00887 or hnRNAP1 induces a splicing switch from PKM2 to PKM1 in BCSCs, triggering a potential therapeutic strategy to selectively suppress PKM2-driven glycolysis in BRCA. hnRNPA1 could serve as an oncofetal gene that is highly upregulated in a various types of cancer, such as pancreatic cancer 34 , 35 , gastric cancer 36 and chronic myelogenous leukemia 37 . Here, we showed that hnRNPA1 is significantly overexpressed in BCSCs than non-BCSCs. Our functional study revealed that hnRNPA1 is required for the maintenance and self-renewal of BCSCs, clarifying the function of hnRNPA1 as oncogenic in BRCA. Previous studies showed that hnRNPA1 acts as a splicing factor, mediating PKM splicing. Consistently, we found that hnRNPA1 binds to the flanking sequences of PKM pre-mRNA exon 9, facilitating the inclusion of exon 10 and the production of PKM2 isoform in BCSCs. This regulation underscores the intricate control mechanisms that govern isoform switching and metabolic adaptation in cancer cells. By interacting with PKM pre-mRNA, hnRNPA1 ensures a preferential splicing outcome that aligns with the metabolic needs of BCSCs. This splicing regulation is critical for maintaining the glycolytic flux and the anabolic processes essential for BCSCs' self-renewal and proliferation. LINC00887 has been investigated in several malignancies, with seemingly contradictory findings regarding its function. Tous et al. revealed that LINC00887 was highly expressed in thyroid Cancer and promoted cell proliferation and invasion 38 . However, LINC00887 has also been reported to be serving as a tumor suppressor in ovarian high-grade serous carcinoma 39 , cervical cancer 40 , and lung cancer 41 . Our study has unveiled an upregulation of LINC00887 levels in BRCA compared to their counterparts. Further analysis revealed that LINC00887 is expressed at a significantly higher level in BCSCs than in non-BCSCs. We also showed that KD of LINC00887 inhibited cell proliferation and induced apoptosis in BRCA cells. Furthermore, our in vitro and in vivo functional studies revealed that LINC00887 maintains the self-renewal of BCSCs by promoting PKM2 expression and reprogramming glucose metabolism. hnRNPA1 can be post-translationally modified by ubiquitination, which mediates its stability 25 , 26 , 42 , 43 . In this study, we identified that LINC00887 upregulates PKM2 by binding to hnRNPA1, thereby concealing its ubiquitination site, preventing its degradation, and ensuring its stability. The stabilization of hnRNPA1 by LINC00887 underscores a layer of post-translational regulation that fine-tunes the expression of metabolic enzymes in response to cellular and environmental cues. In summary, our study demonstrated the strong dependency of LINC00887 for the self-renewal of BCSCs. We also revealed a previously unappreciated signaling involving LINC00887, ubiquitination, hnRNPA1, and PKM splicing, which controls aerobic glycolysis and exerts its critical role in the pathogenesis of BRCA, cancer metabolism, and BSCSs self-renewal. Considering the essential roles of LINC00887 in BRCA pathogenesis and the maintenance of BCSCs, targeting LINC00887 LINC00887/hnRNPA1/PKM2 signaling holds great therapeutic potential for the treatment of BRCA patients by eliminating BCSCs. Our work provides novel insights into the combination of epigenetic-modulating agents and cancer metabolism-targeting agents for cancer therapy. Materials and Methods Clinical samples In the current study, we included 136 paired BRCA tissues and matched adjacent-tumor controls obtained from the Department of General Surgery of the First Hospital of China Medical University and the Department of Medical Oncology of Cancer Hospital of China Medical University between March 2013 and December 2017. This study was approved by the Medical Ethics Committee of China Medical University, and all patients signed written informed consent forms before enrollment. The tissues were immediately snap-frozen in liquid nitrogen after separation and stored at -80°C until use. Information on clinicopathological parameters can be found in Table S1 . Microarray analysis Total RNA was extracted from cells using TRIzol reagent according to the manufacturer’s instructions (15596026, Invitrogen). RNA samples were subjected to GeneChip® Human Transcriptome Array 2.0 (HTA2.0, Affymetrix, USA), which includes global profiling of full-length transcripts. The cDNA labeling, microarray hybridization and data acquisition were performed according to the manufacturer’s instructions. Feature Extraction software (version10.7.1.1, Agilent Technologies) was used to extract raw data from microarray images. Genespring software (version 13.1; Agilent Technologies) was employed for quantile normalization and background correction. Gene Set Enrichment Analysis (GSEA) with differentially expressed genes was performed to identify enriched pathway. Differentially expressed genes between BCSCs and non-BCSCs were identified with Log 2 Fold Change ≥ 1.0 and P ≤ 0.05. The raw data have been deposited in NCBI’s Gene Expression Omnibus (GEO) under accession number GSE273726. Cell culture Human breast cancer cell lines including MCF-7 (ATCC cat.HTB-22, RRID: CVCL_0031) and T47D (ATCC cat.HTB-133, RRID: CVCL_0553) were obtained from the American Type Culture Collection (ATCC). T47D were cultured in DMEM supplemented with 10% FBS; MCF7 was maintained in DMEM supplemented with 10% FBS and 0.01 mg/ml insulin (I8830, Solarbio). Mammospheres were cultured as reported previously by He et al 44 . Briefly, MCF7 sphere (MCF7 MS) and T47D sphere (T47D MS) were cultured in suspension in serum-free DMEM-F12 supplemented with 2% B27 (17504044, Invitrogen), 20 ng/ml human recombinant EGF (AF-100-15, Peprotech), 10 ng/ml human recombinant basic FGF (100-18B, Peprotech). Cells grew under these conditions as nonadherent spheroid cell masses, named mammospheres (MS). Penicillin-Streptomycin (15140122, Gibco) and Plasmocin prophylactic (ant-mpp, InvivoGEN) were supplemented to all the media to prevent potential contamination. Lentivirus infection Lentiviral particles for shCtrl, sh887#1, sh887#2, GV146-LINC00887 and PCDH-hnRNPA1 were purchased from Shanghai Genechem Co., Ltd. Sh887 sequence is seen in Supplementary Table S2 . On the first day, 2×10^5 cells were seeded in 6-well plates. The following day, lentivirus (shCtrl,MOI = 10; sh887, MOI = 20) was directly added to the cells in the presence of 5 µg/ml polybrene (H9268, Sigma-Aldrich). Lentivirus were incubated with the cells for 24 hours, after which the medium was replaced with fresh medium. 48 hours after infection, the infected cells were subjected to puromycin (2µg/ml) select stably transfected cell lines (P8833, Sigma-Aldrich). This screening procedure was repeated 3–5 times until all cells expressed green fluorescent protein. RT-PCR assay and qPCR assay Total RNA was extracted from cells using TRIzol reagent according to the manufacturer’s instructions (15596026, Invitrogen). Reverse transcriptase reaction was performed with 500–1000 ng of total RNA in a total reaction volume of 20 µL using HiScript III RT SuperMix for qPCR (+ gDNA wiper) (R323-01, Vazyme). Quantitative real-time PCR (qPCR) was performed with ChamQ SYBR qPCR Master Mix (Low ROX Premixed) (Q331-02, Vazyme) on the QuantStudio 7 Flex PCR system (Applied Biosystems). Target gene expression levels were normalized to β-actin. Each reaction was performed three times, and relative mRNA abundance was calculated using the contrast ΔΔCT method. The calculated mRNA abundance of glycolytic enzyme genes was further transformed by Z-score normalization to create a heat map. All primers used for qPCR analysis were ordered from Sangon Biotech (Shanghai, China). The primers used for qPCR analysis were listed in Table S3. Protein extraction and Western blot analysis For protein extraction, cells were collected and washed twice with chilled PBS. Total proteins were extracted with RIPA buffer (P0013B, beyotime) containing protease inhibitor and phosphatase inhibitor cocktail (P1050, beyotime). The separation of nuclear and cytoplasmic fractions was performed using the PARIS™ Kit (Thermo Fisher) according to the manufacturer's instructions. Protein concentration was determined by BCA Protein Assay Kit (P0012, beyotime). Protein lysates were diluted with 5X SDS-PAGE Sample Loading Buffer (P0015L, beyotime) and denatured at 99℃ for 10min. For western blot, equal amounts of proteins were separated by 10% SDS-PAGE gels and transferred to polyvinylidene fluoride (PVDF) membranes (IPVH00010, Millipore). Then, the membranes were blocked with 5% milk and incubated with specific primary antibodies overnight at 4℃. Finally, membranes were incubated with secondary antibody and visualized by immunoblotting with Pierce ECL Western blotting Substrate (32106, Thermo Fisher). Primary antibodies used in this study were as follows: PKM1 (1:3000; 15821-1-AP, Proteintech), PKM2 (1:3000; 15822-1-AP, Proteintech), hnRNPA1 (1:2000; Cat. # 8443, Cell signaling technology), CD44 (1:1000; ab157107, Abcam), PARA (1:1000, Cat. # 9542T, Cell signaling technology), β-actin (1:5000; 66009-1-Ig, Proteintech) and GAPDH (1:5000, 60004-1-Ig, Proteintech). Tissue microarray (TMA) and immunohistochemistry (IHC) TMA and IHC assays were performed as previously described 45 . Briefly, BRCA tissue and matched tumor-adjacent control samples in paraffin blocks were visualized using hematoxylin and eosin-stained slides. Tissue cores with a diameter of 1.5 mm were extracted from each paraffin block and precisely arranged into a new paraffin block containing multiple cores using tissue microarray technology (Pathology Equipment). Each TMA section (4 µm) was deparaffinized in xylene and rehydrated in a graded ethanol series. The sections were then incubated overnight at 4°C with specific primary antibodies against PKM1(15821-1-AP,1:200, Proteintech, USA), PKM2(15822-1-AP,1:200, Proteintech, USA), hnRNPA1(15821-1-AP,1:1000, Proteintech, USA),CD44((960-MSM1-P0, 1:100; Thermo Fisher Scientific, USA), CD24(MA5-11828,1:200, Thermo Fisher Scientific, USA).The sections were subsequently incubated with a biotinylated secondary antibody at 37°C for 30 minutes. Horseradish peroxidase-coupled streptavidin (Dako, Glostrup) was then added and incubated for 30 minutes, followed by DAB (3,3-diaminobenzidine) staining. Finally, the sections were counterstained with hematoxylin and mounted with neutral resin. Protein expression levels were observed and counted under a microscope (Eclipse 8i, Nikon). Immunoreactivity was evaluated independently by two investigators according to the percentage of stained cells and staining intensity. Staining intensity was categorized as 0 (absent), 1 (weak), 2 (moderate), or 3 (strong). The final score was calculated by multiplying the intensity score by the percentage score of positively stained cells, yielding a total range of 0–300. To establish an expression-level cutoff (low vs. high), receiver operating characteristic (ROC) curve analysis was applied, with the optimal cutoff determined by maximizing the area under the curve (AUC) and minimizing the sum of sensitivity and 1-specificity for clinical variables. LncRNA in situ hybridization (ISH) assay RNA ISH was performed with Enhanced Sensitive ISH Detection Kit Ⅰ (POD) (MK1030, Boster) according to the manufacturer’s instructions. After deparaffinization and deproteinization, the sections were pre-hybridized in pre-hybridization solution at 42°C for 2 hours. The sections were then incubated with dig-labeled probe solution at 37°C overnight. Following stringent washing, the slides were exposed to a streptavidin-peroxidase reaction system and stained with DAB (ZLI-9017, Zsbio) for 2 minutes. Subsequently, 0.1% hematoxylin (H8070, Solarbio) was used to counterstain the slides for 5 minutes. LINC00887 expression levels were observed and counted under a microscope (Nikon, Tokyo, Japan). The expression score for LINC00887 was calculated as described above of ICH. The LINC00887 probe is shown in Table S4. Cell proliferation assay Cell proliferation was assessed by Cell Counting Kit-8 (CCK-8) assay (341–07761, Dojindo) following the manufacturer’s instructions. Briefly, cells infected with indicated lentivirus were seeded on 96-well plates in triplicates at a density of 3000 cells/well in a final volume of 100 ml. Then, 10 µl CCK-8 solution was added to each well at indicated time points and incubated at 37°C for 2–4 h. Finally, the absorbance at 450 nm was determined using an Anthos 2010 microplate reader (Anthos Labtec Instruments GmbH, Austria). Semi-quantitative RT-PCR and PKM splicing assays PKM splicing assays were performed as previously described 16 . Briefly, total RNA was extracted from cells using TRIzol reagent. Semi-quantitative RT-PCR were used to analyze alternative spliced products. The PCR products of PKM were digested with FastDigest PstI endonuclease (Thermo, Scientific). Finally, the digested products were resolved by 6% non-denaturing PAGE and images were taken by BioSpectrum AC Imaging System (UVP). The primers for PKM variants are shown in Table S3. In vivo limiting dilution assay For in vivo limiting dilution assay, 3 x 10 4 , 3 x 10 5 , 3 x 10 6 cells control or LINC00887 KD MCF7 MS were injected subcutaneously into BALB/c nude mice. The number of recipient mice developed tumor within 20 days post-transplantation was counted for each group with each dose of donor cells. The frequency of BCSCs were estimated by ELDA software ( http://bioinf.wehi.edu.au/software/elda/ ). Soft-agar colony formation assay Soft agar colony formation assay was carried out as described previously 46 . Briefly, 1.2% agarose gel (50004, Lonza Rockland) was mixed with phenol red-free DMEM medium supplemented with 20% FBS and 2% penicillin-streptomycin in 6-well plates as the bottom layer. Mix the cells (10 3 cells/ml) into 0.6% agarose gel and phenol red‐free DMEM medium (1:1) supplemented with 20% FBS and 2% penicillin-streptomycin as the top layer. An additional 600 µl medium were added to the uppermost layer, and the medium was added every 3–4 days. After 2–3 weeks of incubation at 37°C with 5% CO 2 , the colonies larger than 150 µm in diameter were counted with MTT (5mg/ml) staining. Spheroid formation assay Mammospheres cells (2000cells/ml) were collected and re-suspended in DMEM/F-12 supplemented with 2% B27 (17504044, Invitrogen), 20 ng/ml human recombinant EGF (AF-100-15, Peprotech), 10 ng/ml human recombinant basic FGF (100-18B, Peprotech) in 6-well Ultra-Low Attachment Microplates plates (3473, Corning™). 0.5 mL of fresh medium was added to each well every 3 days. After 2 weeks of incubation at 37°C in a humidified environment with 5% CO 2 , the number of spheres > 100 µm in diameter was counted under an inverted microscope (Nikon TE2000-U, Japan). Flow cytometry assays The cells were harvested, washed with chilled PBS 3 times, resuspended in 100ul Flow Cytometry staining buffer (00-4222-26, eBioscience) containing APC-conjugated CD44 antibody (103011, BioLegend) and PE-conjugated CD24 antibody (311105, BioLegend) and incubated at 4°C for 30 min in the dark. After incubation, the cells were washed with PBS 3 times. Then, single-cell suspensions were subjected to analysis by MACSQuatTM Flow cytometer (Becton-Dickinson, USA) within 2 h. RNA pull-down assay RNA pull-down was performed with Pierce™ Magnetic RNA Protein Pull Down Kit (20164, Thermo Fisher Scientific) following the manufacturer’s instructions. Briefly, biotin-labeled probes were conjugated to Streptavidin agarose resin beads (Thermo Fisher Scientific). Then, biotin-labeled probe-conjugated streptavidin beads were incubated with nuclear extracts in Protein-RNA binding buffer overnight at 4°C. After washed 4 times with 1× washing buffer, the protein-RNA-beads mixture was dissolved in 1× SDS buffer and analyzed by western blot. The LNC887 and PKM probes are shown in Table S5. RNA Immunoprecipitation and Semi-quantitative RIP was performed as described previously with some modifications 47 . Briefly, cells were collected, washed with chilled PBS, and lysed with 1ml Co-IP lysis buffer (PR20037, Proteintech) with protease inhibitor and RNase inhibitor. The lysates were centrifuged at 12,000 g for 10 min at 4°C. Then, supernatants were collected. 5% of lysate was saved as input. HnRNPA1 antibody (11176-1-AP, Proteintech) and IgG antibody (30000-0-AP, Proteintech) were conjugated to Protein A/G Magnetic Beads by rotation at 4°C for 4 h. Supernatants were incubated with the antibody-conjugated beads with rotation at 4°C overnight. Afterward, beads were washed 3 times with chilled RIP buffer and followed by Proteinase K (E00492, Thermo Fisher Scientific) treatment at 55°C for 1h. The input and co-immunoprecipitated RNA were extracted with TRIzol reagent and analyzed by Semi-quantitative RT-PCR. Lactate Levels Lactate levels were determined with CheKine™ Micro Lactate Assay Kit (KTB1100, Abbkine) following the manufacturer’s instructions. Briefly, the cell supernatant was harvest, filtered with 5 kDa MWCO Amicon® Ultra Centrifugal Filter (UFC901008, Millipore) for deproteinization. The deproteinized samples were then added to a 96-well plate in triplicates with appropriate dilution and incubated with the lactate reaction mix at 37°C for 30 minutes in the dark. The absorbance was measured at a wavelength of 450 nm and the lactate levels were calculated with a standard curve. Measurements of ECAR Using the XFe24 extracellular flux analyzer ECAR was determined with a Seahorse XFe24 Extracellular Flux Analyzer and a Seahorse XF Glycolytic Rate Assay Kit (102342-100, Agilent Technologies) according to the manufacturer’s instructions. In brief, 1.5 × 10 4 cells per well were seed in XF24 cell plates. Cells were cultured in XF RPMI medium (without phenol red) supplemented with 2 mM glutamine, 10 mM glucose, 1 mM pyruvate, and 5 mM HEPES in a CO2-free incubator for 1h prior to the assay. The glycolytic rates were analyzed using a Seahorse Bioscience extracellular flux analyzer (Seahorse XF24 FluxPak, Agilent Technologies). Cycloheximide (CHX) treatments After MCF-7 cells were transfected with the overexpressed 887 plasmid, cells were treated with CHX (HY-12,320, MCE), and then the protein was collected at 0h,3h and 5h, respectively WB analysis was performed. Xenograft model Four-week-old female BALB/c-nu mice were purchased from the Hua Fukang Biological Technologies Inc. The mice were acclimated to the new environment for one week before the experiment. Prior to tumor cell inoculation, they were randomly divided into groups with approximately equal numbers. The researcher conducting the experiment was blinded to the grouping of the animals. A total of 3 x 10^6 MCF7 MS were injected subcutaneously into the right armpit region of each mouse. All protocols followed the Regulations of Experimental Animal Administration issued by the Ministry of Science and Technology of the People's Republic of China. Once the tumors became visible, their weight and size were measured every five days. Body weight and tumor growth were also assessed every five days. Tumor number or tumor weight in each group was assessed in a blinded manner. Tumor volume was calculated using the equation V = 0.5 × D × d^2, where V represents the tumor volume, D is the longitudinal diameter, and d is the latitudinal diameter. Thirty days after injection, the mice were sacrificed, and the subcutaneous tumors were isolated and measured. Additionally, tumor tissue was fixed in 10% formalin for further experiments. Statistical analysis All statistical analyses were performed using the SPSS 26.0 software package (SPSS Inc., Chicago, USA) and GraphPad Prism 8 software (GraphPad, USA). A P-value of less than 0.05 was considered statistically significant. Pearson's χ2 or Fisher's exact test was employed to determine the associations between indicator expression and clinicopathological parameters in BRCA patients. Linear regression analysis was used to analyze the correlation of LINC00887 with other indicators. Student's t-test (two-tailed), Wilcoxon's test, or Welch's t-test were used to compare significant differences between groups for paired and unpaired continuous variables, presented as mean ± standard deviation (s.d.) or median (quartile). The Kaplan-Meier method and Log-rank test were used to estimate prognosis. For cell experiments, the sample size was determined to be adequate based on the magnitude and consistency of measurable differences between groups, typically three or more samples. For the xenograft mouse experiments, no statistical method was used to predetermine the sample size, which was based on previous experimental observations. The sample size for each experiment is shown in the figure legend. No data were excluded from the analysis. Declarations Availability of data and materials The datasets used and/or analyzed in this article were included within the article and the additional files. Please contact the corresponding author for data requests. ACKNOWLEDGEMENTS This study was supported by National Natural Science Foundation of China (NSFC, No.82473134, No.81902708, No.82400216, China), Liaoning Province Higher Education Scientific Research Funding Project (No.JYTM20230128, No.2023JH2/2020008, China), Science and technology innovation team project of China Medical University (No.CXTD2022007, China), "Xingliao Talent Program" of Liaoning Province (XLYC2403200, China). Liaoning Province Science and Technology Plan Project - Special Project on Technological Innovation Facilitating the High-Quality Development of China Medical University( 2025JH2/102800061). AUTHOR CONTRIBUTIONS X. Lv: Conceptualization, resources, formal analysis, supervision, validation, investigation and writing–original draft. L. Han: Conceptualization, resources, funding acquisition, methodology and writing–original draft. W. Tong: Conceptualization, resources, formal analysis, supervision. X. Sun: Methodology. Y. Yan: Methodology, funding acquisition. S. Zhou: Methodology, validation. S. Xu: Methodology, validation. D. Zhang: Methodology, validation. J. Wang: Methodology, validation. J. Liu: Methodology, validation. Y. Zhang: Methodology, validation. H. Zhao: Methodology. W. Yao: Methodology. M. Wei: Conceptualization, data curation, supervision, funding acquisition, project administration, writing–review and editing. B. Chen: Conceptualization, resources, methodology, and project administration. M. He: Conceptualization, data curation, supervision, funding acquisition, project administration, writing–review and editing. ETHICS APPROVAL AND CONSENT TO PARTICIPATE All animal experiments were performed in accordance with the relevant institutional and national guidelines and the regulations of the Medical Laboratory Animal Health Committee of China Medical University (CMU20241515). This study was approved by the Clinical Research Ethics Committee of China Medical University (No. 2019080). CONSENT FOR PUBLICATION Not applicable. 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Supplementary Files FigS2.tif Supplemental Fig 2 LINC00887 Mediates BRCA Progression (A) Full Sequence Map for GV248-shLINC00887. (B) qPCR analysis showing KD efficiency of LINC00887 shRNAs in MCF-7 MS and T47D MS. (C) Ectopic expression of LINC00887, as detected by RT-qPCR. (D) qPCR analysis showing overexpression efficiency of LINC00887 in MCF-7 and T47D. (E) Effect of LINC00887 KD on cell proliferation in MCF-7 MS and T47D MS. (F) Effect of LINC00887 KD on spheroid formation in MCF-7 MS and T47D MS. (G) Effect of LINC00887 KD on the proportion of CD44 + CD24 - BCSCs. (H) Effect of LINC00887 OE on colony formation in MCF-7 cells. (I) Representative micrographs showing effect of LINC00887 overexpression on mammosphere formation. (J) Representative xenograft tumor images. (K) Growth curves of MCF7 MS xenograft tumors transduced with LINC0087 shRNA. (L) Diagram of the glycolysis pathway. (M) PKM1 (left) and PKM2 (right) expression in BRCA tissue with TNM stage. (N) ROC curves illustrate the performance of employing PKM1 expression (left) and PKM2 expression (right) to predict tumor stages in our in-house BRCA cohort. Data were shown as mean ± s.d. of at least three independent experiments. Data were shown as mean ± s.d. *P < 0.05; **P < 0.01; ***P < 0.001. FigS3.tif Supplemental Fig 3 HnRNPA1 is Upregulated in BCSCs and Correlates with poor prognosis (A) The volcano plot of differentially expressed splicing factors between MCF-7 and MCF-7 MS. (B) The 3D interaction diagrams for QKI protein and PKM pre-mRNA. (C) Violin charts displaying the expression levels of hnRNPA1 in BRCA tissue and normal control. (D) IHC assay was performed to determine the protein expression of hnRNPA1 in BRCA vs normal control with our in-house BRCA cohort. The scale bar represents 50 μm. (E) The expression levels of hnRNPA1 in BRCA vs normal control in the TCGA cohort. (F) Analysis of the relationship between hnRNPA1 protein expression and prognosis in BRCA by KM plotter. (G-I) ROC curves illustrate the performance of employing hnRNPA1 expression to predict tumor stages (G), tumor size (H) and lymphatic metastasis (I) in our in-house BRCA cohort. ***P < 0.001. FigS4.tif Supplemental Fig 4. LINC00887 Mediates Aerobic Glycolysis through hnRNPA1/PKM2 Axis (A) Relative levels of PKM1 and PKM2 in MCF-7 MS after transduction with indicated lentiviruses. (B) Radar chart shows glycolytic rates at different stages in MCF-7 MS after transduction with indicated lentiviruses. Uneditedblotandgelimages.pdf Unedited blot and gel images Supplementalinformation.docx supplementary tables Cite Share Download PDF Status: Published Journal Publication published 14 Apr, 2026 Read the published version in Oncogene → Version 1 posted Editorial decision: revise 29 Oct, 2025 Review # 1 received at journal 26 Oct, 2025 Review # 2 received at journal 22 Oct, 2025 Review # 3 received at journal 17 Oct, 2025 Reviewer # 3 agreed at journal 13 Oct, 2025 Reviewer # 2 agreed at journal 09 Oct, 2025 Reviewer # 1 agreed at journal 05 Oct, 2025 Reviewers invited by journal 02 Oct, 2025 Submission checks completed at journal 29 Sep, 2025 Editor assigned by journal 26 Sep, 2025 First submitted to journal 26 Sep, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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1","display":"","copyAsset":false,"role":"figure","size":11563216,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe High Expression of LINC00887 is Positively Correlated with PKM2-Mediated Glycolysis in BCSCs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Strategy diagram to identify downstream target genes of LINC00887. (B) Top 10 enriched GSEA pathways in the up-regulated genes from MCF-7 MS versus MCF-7. Pathways are arranged by their normalized enrichment score. (C-E) Measurement of glycolysis levels in MCF-7 cells and MCF-7 MS. Graph (C) shows the extracellular acidification rate (ECAR) over time. Volume (D) shows glycolytic rates at different stages. (E) shows the measurement of lactate levels in parental cells and MS. (F) Heatmaps showing top 10 up-regulated LNCRNAs in MCF-7 MS compared with MCF-7 cells. (G) Analysis of the relationship between LINC00887 expression and prognosis in BRCA by KM plotter (http://kmplot.com/analysis/ database). (H) Confirmation of the expression of LINC00887 in MS and its parental cells by qPCR. (I) Violin charts displaying the expression levels of LINC00887 in BRCA and adjacent tissues (n=136). (J) Expression levels of LINC00887 in CD44\u003csup\u003e+\u003c/sup\u003eCD24\u003csup\u003e-\u003c/sup\u003e versus non- CD44\u003csup\u003e+\u003c/sup\u003eCD24\u003csup\u003e-\u003c/sup\u003e BRCA from the TCGA dataset. (K) Schematic representation illustrating the effect of LINC00887 KD on the frequency of stem/initiating cells \u003cem\u003ein vivo\u003c/em\u003e. (L) Table shows the donor cell numbers used for \u003cem\u003ein vivo\u003c/em\u003e limiting dilution assay and the ratios of the recipient mice with tumor (n=6). (M) Effect of LINC00887 KD on the frequency of stem/initiating cells determined by ELDA software. (N) The weights of MCF7 MS xenograft tumors on day 30 post-inoculation (n = 6). (O) GSEA plot showing enriched gene sets of glycolysis in high LINC00887 (LINC00887\u003csup\u003e+\u003c/sup\u003e) versus low LINC00887 (LINC00887\u003csup\u003e-\u003c/sup\u003e) samples. (P) Heatmap showing the expression levels of 11 glycolytic enzymes in MCF7 MS transduced with indicated lentiviruses. Data are represented as Z-score normalized intracellular levels. (Q) Comparison of the expression of PKM2 (left) and PKM1 (right) in BRCA tissues and adjacent tissues (n=136). (R) Correlation between LINC00887 and PKM2 (left) or PKM1 (right) expression in the in-house BRCA cohort. Data were shown as mean ± s.d. *P \u0026lt; 0.05; **P \u0026lt; 0.01; ***P \u0026lt; 0.001 and ns, no significance, p ≥ 0.05.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-7722676/v1/d05a4c4f4a72faf2d9bd1e7e.png"},{"id":93617164,"identity":"19c5878d-0c1e-42af-ba88-70711a577ad2","added_by":"auto","created_at":"2025-10-15 17:06:08","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":10406660,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLINC00887 Promotes the Self-renewal of BCSCs by Activating PKM2-Mediated Glycolysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Schematic representing the proposed mechanism by which LINC00887 enhances BCSC properties through activation of PKM2-mediated glycolysis. (B) Western blotting (left) showing the expression of PKM2 and PKM1 in MCF-7 cells and MCF-7 MS cells. Corresponding quantification of PKM isoform distribution (right). (C) Left panel: protein level changes of PKM2 and PKM1 following silencing or overexpressing LINC00887 in MCF-7 MS and MCF-7 cells, detected by western blotting. Right panel: statistical representation of the relative abundance of PKM1 and PKM2 isoforms. (D) Measurement of ECAR upon LINC00887 KD in MCF-7 MS. (E) Effects of LINC00887 KD on lactate levels in MCF-7 MS. (F) Measurement of ECAR upon overexpressing LINC00887 in MCF-7 cells. (G) Effects of LINC00887 overexpression on lactate levels in MCF-7 cells. (H) Schematic diagram illustrating specific restriction digestion of PKM1/2 exon 10 by the Pst1 enzyme. (I) PKM isoform distribution in MCF-7 and MCF-7 MS cells analyzed by the PKM splicing assay, with the percentage of PKM1 and PKM2 indicated below. (J) PKM1 and PKM2 mRNA levels following LINC00887 KD or overexpression in MCF-7 MS and MCF-7 cells. Data were shown as mean ± s.d. *P \u0026lt; 0.05; **P \u0026lt; 0.01; ***P \u0026lt; 0.001 and ns, no significance, p ≥ 0.05.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-7722676/v1/31a23868f6fb40c51c656be9.png"},{"id":93617167,"identity":"3a1a486f-5787-43b1-95c7-63b28d1f0c19","added_by":"auto","created_at":"2025-10-15 17:06:09","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":12274711,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHnRNPA1 Binding to PKM Pre-mRNA Triggers Mutually Exclusive Splicing\u003c/strong\u003e \u003cstrong\u003etoward PKM2 to Activate Glycolysis in BRCA\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e Schematic illustrating hnRNPA1 binding to PKM pre-mRNA, triggering mutually exclusive splicing to produce PKM2 and activate glycolysis in BRCA. (B) Heatmaps showing differential expression of splicing factors in MCF-7 MS vs. MCF-7 cells. (C)MOE docking analysis revealing binding scores of splicing factors with PKM. (D) The 3D interaction diagrams depicting hnRNPA1 binding to PKM pre-mRNA. (E) Schematic showing hnRNPA1 binding to exon 9 flanking sequences of PKM pre mRNA to trigger PKM2. (F) RNA pulldown assay showing that endogenous hnRNPA1 directly binds with synthetic WT not MUT PKM probes. (G) Effect of hnRNPA1 overexpression on PKM1 and PKM2 mRNA levels in MCF-7 cells. (H) Effects of hnRNPA1 overexpression on protein levels of PKM1 and PKM2 in MCF-7 cells. (I) Statistical analysis depicting the protein level changes of PKM1 and PKM2 following hnRNPA1 overexpression in MCF-7 cells. (J) Pearson’s correlation analysis of hnRNPA1 and PKM2 protein expression levels in our in-house BRCA. (K) GSEA plot showing enrichment of glycolysis-related gene signatures in hnRNPA1⁺ versus hnRNPA1⁻ BRCA from the TCGA database. (L) Measurement of ECAR upon overexpressing hnRNPA1 in MCF-7 cells. (M) The expression of hnRNPA1 in BRCA vs normal control with our in-house BRCA cohort (n=136). (N) Western blot showing the protein expression of hnRNPA1 in parental cells vs MS.（O）Circular plot illustrating the percentage of patients with high/low expression of hnRNPA1 based on various clinicopathological parameters, including Stage I/Stage II\u0026amp;III, tumor size (≤2.5 cm/\u0026gt;2.5 cm), and lymphatic metastasis status. Data were shown as mean ± s.d. *P \u0026lt; 0.05; **P \u0026lt; 0.01; ***P \u0026lt; 0.001 and ns, no significance, p ≥ 0.05.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-7722676/v1/f794831fcdab8c8c95cd49d1.png"},{"id":93618246,"identity":"c6415066-d6f6-4519-aaeb-a4f0f1e75169","added_by":"auto","created_at":"2025-10-15 17:22:09","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":13603774,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLINC00887 Interacts directly with hnRNPA1 to Modulate its Expression, thereby Maintaining the Stemness of BCSCs.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Pearson correlation analysis of LINC00887 and hnRNPA1 protein expression across BRCA samples. (B)The catRAPID heat-map shows the prediction of the interaction between LINC00887 and hnRNPA1 protein (\u003ca href=\"http://www.tartaglialab.com/\"\u003ehttp://www.tartaglialab.com/\u003c/a\u003e). The x- and the y-axes represent the nucleotide index of LINC00887 and the amino acid index of hnRNPA1 protein, respectively. (C) MOE docking platform showing the predicted interaction sites between LINC00887 tertiary structure and hnRNPA1 protein. (D) RIP followed by PCR showed the binding of LINC00887 with hnRNPA1 in MCF-7 MS, with IgG used as a negative control. (E) qPCR analysis of hnRNPA1 mRNA level in LINC00887 KD MCF7 MS. (F) Protein level change of hnRNPA1 in LINC00887 KD MCF7 MS. T: total cell lysates, C: represents cytoplasmic lysates, and N: represents nuclear lysates. (G) qPCR analysis of LINC00887 level upon modulating LINC00887 and hnRNPA1 in MCF-7 MS and T47D MS. (H) Protein level change of hnRNPA1 in MCF-7 MS and T47D MS upon LINC00887 KD with or without hnRNPA1 overexpression, as detected by western blotting. (I) The effects of hnRNPA1 overexpression on cell proliferation in MCF-7 MS and T47D MS upon LINC00887 KD. (J)The effects of hnRNPA1 overexpression on CD44\u003csup\u003e+\u003c/sup\u003e/CD24\u003csup\u003e-\u003c/sup\u003e population in MCF-7 MS and T47D MS upon LINC00887 KD. (K) Effect of hnRNPA1 overexpression on mammosphere formation in MCF-7 MS and T47D MS upon LINC00887 KD. Data were shown as mean ± s.d. *P \u0026lt; 0.05; **P \u0026lt; 0.01; ***P \u0026lt; 0.001 and ns, no significance, p ≥ 0.05.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-7722676/v1/94f48898cb326c1becd9bb28.png"},{"id":93618247,"identity":"fca8a442-297e-4ced-8e85-6d331a0fe91a","added_by":"auto","created_at":"2025-10-15 17:22:09","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":11508965,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLINC00887 mediates the triggering of PKM2 by hnRNPA1 to activate glycolysis by blocking the ubiquitination site of hnRNPA1\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Diagram of the mechanism by which LINC00887 blocks the ubiquitination modification site of hnRNPA1, stabilizing its expression and subsequently upregulating PKM2. (B) Prediction of ubiquitination sites of hnRNPA1 protein by BDM-PUB database (\u003ca href=\"http://bdmpub.biocuckoo.org/\"\u003ehttp://bdmpub.biocuckoo.org/\u003c/a\u003e). (C) RNA pull-down followed by western blot showing the interaction of the WT 887 and Mut 887 probes with hnRNPA1 protein in LINC00887 KD MCF-7 MS. (D) Effect of LINC00887 KD on the ubiquitination level of hnRNPA1 protein. (E) Effect of LINC00887 KD on the stability of hnRNPA1 protein in MCF-7 cells. (F) Quantification of hnRNPA1 protein level shown in (E). (G) Expression of PKM2, PKM1 and CD44 in MCF-7 MS and T47D MS infected with indicated lentiviruses. (H) Lactate levels in MCF-7 MS and T47D MS infected with indicated lentiviruses. (I) Glycolytic rate analysis in LINC00887 KD MCF-7 MS with or without hnRNPA1 overexpression. Data were shown as mean ± s.d. *P \u0026lt; 0.05; **P \u0026lt; 0.01; ***P \u0026lt; 0.001 and ns, no significance, p ≥ 0.05.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-7722676/v1/a68524a3249e44c277cf9c0a.png"},{"id":93619043,"identity":"93685a32-35d7-4283-b645-e7e3d8e9f222","added_by":"auto","created_at":"2025-10-15 17:30:09","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":19020070,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLINC00887/hnRNPA1/PKM2 mediates BRCA progression \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vivo\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Diagram illustrating \u003cem\u003ein vivo\u003c/em\u003e xenograft tumorigenesis assays with human MCF7 MS and nude mice. (B) Representative \u003cem\u003ein vivo\u003c/em\u003e bioluminescent images of nude mice subcutaneously inoculated with MCF7 MS transduced with indicated lentiviruses on day 5 and day 30 post-inoculation. (C) Growth curves of MCF7 MS xenograft tumors transduced with indicated lentiviruses (n = 6). (D) Representative xenograft tumor images. (E) Kaplan-Meier survival curves of nude mice subcutaneously inoculated with MCF7 MS transduced with indicated lentiviruses (n = 6). (F) The weights of MCF7 MS xenograft tumors on day 30 post-inoculation (n = 6). (G) qPCR assay was performed to determine the expression of LINC00887 in the indicated groups. (H-I) IHC assay was performed to determine the protein expression and the ratio of PKM1 and PKM2 in the indicated group. Quantification of the PKM1/PKM2 ratio is shown in (I). (J-K) The protein expression of hnRNPA1, PKM2, PKM1 and CD44 in xenograft tumor tissues for indicated group were determined by western blot. (L) Effect of hnRNPA1 on glycolytic rates in LINC00887 KD MCF7 MS xenograft tumors. Data were shown as mean ± s.d. of at least three independent experiments. Data were shown as mean ± s.d. *P \u0026lt; 0.05; **P \u0026lt; 0.01; ***P \u0026lt; 0.001 and ns, no significance, p ≥ 0.05.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-7722676/v1/0937e1a71aaec8a817f98d39.png"},{"id":106949727,"identity":"62e1d419-3f9a-4644-821a-1f75b04a52c8","added_by":"auto","created_at":"2026-04-15 07:14:32","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":76728036,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7722676/v1/1a142cbf-303a-4b63-8a49-0778f200547c.pdf"},{"id":93617174,"identity":"59dc7ee3-2beb-4e49-b6c2-9cef3cf3eb66","added_by":"auto","created_at":"2025-10-15 17:06:09","extension":"tif","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":13917520,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplemental Fig 2 LINC00887 Mediates BRCA Progression\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A)\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; Full Sequence Map for GV248-shLINC00887. (B) qPCR analysis showing KD efficiency of LINC00887 shRNAs in MCF-7 MS and T47D MS. (C) Ectopic expression of LINC00887, as detected by RT-qPCR. (D) qPCR analysis showing overexpression efficiency of LINC00887 in MCF-7 and T47D. (E) Effect of LINC00887 KD on cell proliferation in MCF-7 MS and T47D MS. (F) Effect of LINC00887 KD on spheroid formation in MCF-7 MS and T47D MS. (G) Effect of LINC00887 KD on the proportion of CD44\u003csup\u003e+\u003c/sup\u003eCD24\u003csup\u003e-\u003c/sup\u003e BCSCs. (H) Effect of LINC00887 OE on colony formation in MCF-7 cells. (I) Representative micrographs showing effect of LINC00887 overexpression on mammosphere formation. (J) Representative xenograft tumor images. (K) Growth curves of MCF7 MS xenograft tumors transduced with LINC0087 shRNA. (L) Diagram of the glycolysis pathway. (M) PKM1 (left) and PKM2 (right) expression in BRCA tissue with TNM stage. (N) ROC curves illustrate the performance of employing PKM1 expression (left) and PKM2 expression (right) to predict tumor stages in our in-house BRCA cohort. Data were shown as mean ± s.d. of at least three independent experiments. Data were shown as mean ± s.d. *P \u0026lt; 0.05; **P \u0026lt; 0.01; ***P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"FigS2.tif","url":"https://assets-eu.researchsquare.com/files/rs-7722676/v1/fcd8bd24a587a04b8d113bfb.tif"},{"id":93617176,"identity":"4d3f53d6-9289-408e-ba78-21c27952c375","added_by":"auto","created_at":"2025-10-15 17:06:09","extension":"tif","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":5695584,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplemental Fig 3 HnRNPA1 is Upregulated in BCSCs and Correlates with poor prognosis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) The volcano plot of differentially expressed splicing factors between MCF-7 and MCF-7 MS. (B) The 3D interaction diagrams for QKI protein and PKM pre-mRNA. (C) Violin charts displaying the expression levels of hnRNPA1 in BRCA tissue and normal control. (D) IHC assay was performed to determine the protein expression of hnRNPA1 in BRCA vs normal control with our in-house BRCA cohort. The scale bar represents 50 μm. (E) The expression levels of hnRNPA1 in BRCA vs normal control in the TCGA cohort. (F) Analysis of the relationship between hnRNPA1 protein expression and prognosis in BRCA by KM plotter. (G-I) ROC curves illustrate the performance of employing hnRNPA1 expression to predict tumor stages (G), tumor size (H) and lymphatic metastasis (I) in our in-house BRCA cohort. ***P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"FigS3.tif","url":"https://assets-eu.researchsquare.com/files/rs-7722676/v1/eb27d00c921484bf78aba8a4.tif"},{"id":93617909,"identity":"92c8e044-d1a7-41ee-9bee-0d9d85f9b546","added_by":"auto","created_at":"2025-10-15 17:14:09","extension":"tif","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":1180872,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplemental Fig 4. LINC00887 Mediates Aerobic Glycolysis through hnRNPA1/PKM2 Axis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Relative levels of PKM1 and PKM2 in MCF-7 MS after transduction with indicated lentiviruses. (B) Radar chart shows glycolytic rates at different stages in MCF-7 MS after transduction with indicated lentiviruses.\u003c/p\u003e","description":"","filename":"FigS4.tif","url":"https://assets-eu.researchsquare.com/files/rs-7722676/v1/7be21d3264a0a0710e63c3ca.tif"},{"id":93619040,"identity":"b1c30cd5-6234-4e2a-a17e-f4338ffaaede","added_by":"auto","created_at":"2025-10-15 17:30:09","extension":"pdf","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":3710124,"visible":true,"origin":"","legend":"Unedited blot and gel images","description":"","filename":"Uneditedblotandgelimages.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7722676/v1/e6af43b0b370a51f6cfaeee1.pdf"},{"id":93617169,"identity":"1ec75a2d-3366-4d92-8229-cbfae8b95753","added_by":"auto","created_at":"2025-10-15 17:06:09","extension":"docx","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":26699,"visible":true,"origin":"","legend":"supplementary tables","description":"","filename":"Supplementalinformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-7722676/v1/d3841e053b3635b3ac28bd88.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e conflict of interest to disclose.","formattedTitle":"Blocking ubiquitination of hnRNPA1 maintains the self-renewal of breast cancer stem cells via mutually exclusive splicing of PKM pre-mRNA","fulltext":[{"header":"Introduction","content":"\u003cp\u003eBreast cancer (BRCA) represents a heterogeneous disease, characterized by diverse subtypes and clinical response\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Within this complexity, research on cellular hierarchies and intratumor heterogeneity provides support for the existence of breast cancer stem cells (BCSCs), characterized by unlimited self-renewal/repopulating potential, which contribute to the initiation and progression of the disease, as well as to treatment failure and recurrence of BRCA\u003csup\u003e\u003cspan additionalcitationids=\"CR4 CR5 CR6 CR7\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Targeting and eliminating BCSCs is considered a promising strategy for BRCA treatment. However, current therapeutic strategies inadequately target BCSCs. Therefore, understanding the biology and regulation of BCSCs holds significant implications for developing more effective therapeutic strategies targeting this resilient cell population.\u003c/p\u003e\u003cp\u003eReprogramming of cellular metabolism is widely considered a hallmark of cancer\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. In contrast to normal healthy cells, tumor cells predominantly rely on glycolysis even in the presence of oxygen, which is termed as \"aerobic glycolysis\" or the \"Warburg effect\"\u003csup\u003e11\u003c/sup\u003e. Emerging evidence highlights that BCSCs exhibit distinct metabolic features, especially enhanced glycolytic activity, which enables them to adapt to nutrient scarcity and contribute to their survival and aggressiveness\u003csup\u003e\u003cspan additionalcitationids=\"CR13\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Pyruvate kinase muscle isozymes (PKMs) serve as rate-limiting enzymes in glycolysis, which catalyzes the conversion of phosphoenolpyruvate (PEP) to pyruvate. Pyruvate kinase muscle isozymes M1 (PKM1) and M2 (PKM2), are derived from mutually exclusive splicing of exons 9 and 10 of PKM gene, with PKM1 including exon 9 and PKM2 including exon 10. It is reported that a splice isoform switch from PKM1 to PKM2 is essential for the transition of cellular metabolism towards aerobic glycolysis, thereby fostering tumorigenesis\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Alternative splicing, a critical mechanism in gene expression, relies on a complex array of splicing factors. Evidence shows that heterogeneous nuclear ribonucleoprotein (hnRNP) proteins, including hnRNPA1, and hnRNPA2 are involved in promoting PKM2 splicing\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Nevertheless, the regulatory mechanisms underlying PKM2 splicing in BCSCs remain elusive.\u003c/p\u003e\u003cp\u003eAs one of the most prevalent post-translational modifications, ubiquitination fine-tunes the fate and function of substrate proteins by regulating their stability, localization, activity, and interactions with other proteins\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Long noncoding RNAs (LNCRNAs) are versatile regulators of gene expression with functions spanning various levels of cellular regulation, from transcriptional and post-transcriptional processes to chromatin organization and cell signaling \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Evidence reveals that LNCRNAs play multifaceted roles in regulating protein ubiquitination, as they bind directly to proteins, block ubiquitination sites, modulate enzyme activity, and act as scaffolds or guides for protein complexes involved in ubiquitination\u003csup\u003e\u003cspan additionalcitationids=\"CR20 CR21\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Dysregulation of LNCRNAs is closely associated with various cancer types, including breast cancer\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Previous studies have revealed that hnRNPs undergo ubiquitination and subsequent proteasomal degradation\u003csup\u003e\u003cspan additionalcitationids=\"CR26\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. However, whether LNCRNAs regulate the ubiquitination of hnRNPs has yet to be addressed.\u003c/p\u003e\u003cp\u003eIn this work, we performed a genome-wide expression profiling in BCSCs and identified LINC00887 as a key regulator of BRCA stemness. We showed that the expression of hnRNPA1 was markedly upregulated in BCSCs, where it binds to the flanking sequence of PKM pre-mRNA exon 9, inducing a switch to the PKM2 isoform, a crucial requirement for the self-renewal of BCSCs. Furthermore, we identified that LINC00887 prevents ubiquitination-mediated hnRNPA1 degradation to enhance aerobic glycolysis by regulating mutually exclusive splicing of PKM pre-mRNA. These data highlight targeting cancer metabolism as a promising therapeutic strategy for BRCA.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eThe high expression of LINC00887 is positively correlated with PKM2-mediated glycolysis in BCSCs\u003c/h2\u003e\u003cp\u003eTo explore the pathogenesis of BCSCs, we utilized serum-free suspension culture to enrich the subpopulation of CD44\u003csup\u003e+\u003c/sup\u003e/CD24\u003csup\u003e\u0026minus;\u003c/sup\u003e MCF-7 mammospheres (MCF-7 MS) with BCSC-like properties, then performed microarray on MCF-7 MS along with its parental MCF-7 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). We identified thousands of dysregulated genes in MCF-7 MS (Supplementary Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA). Notably, gene set enrichment analyses (GSEA) revealed significant enrichment for glycolysis in MCF-7 MS (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). To further corroborate this discovery, we detected glycolytic rates by XF glycolytic stress assays, as well as determining lactate levels in MS and its parental cells. Consistently, MS showed elevated glycolytic rates and higher lactate levels compared to its parental cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC-\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). These findings strongly suggest that BCSCs exhibit active aerobic glycolysis, heavily relying on glycolytic metabolism to sustain their energy requirements and other vital biological functions.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003ePrior investigations into the transcriptomic diversity of BCSCs have primarily focused on mRNA, little is known about LNCRNA heterogeneity. To identify the LNCRNAs involved in the maintenance of BCSCs properties, we performed genome-wide LNCRNA microarray on MCF-7 MS along with its parental MCF-7 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Principal component analysis (PCA) and cluster analysis showed that the MCF-7 MS group could be separated from the parental MCF-7 cells group, indicating a distinct LNCRNA signature (Supplementary Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eB). Further analysis of microarray data identified hundreds of differentially expressed LNCRNAs, including 759 down-regulated and 632 up-regulated LNCRNAs in MCF-7 MS compared to parental MCF-7 cells (Supplementary Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eC). We focused on the top 10 MCF-7 MS-enriched LNCRNAs to determine prognostic LNCRNAs in breast cancer (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF), found that both the expression of LINC00887 and MGC27382 significantly correlate with poorer survival in breast cancer patients (n\u0026thinsp;=\u0026thinsp;1538, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://kmplot.com/analysis/\u003c/span\u003e\u003cspan address=\"http://kmplot.com/analysis/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG and Supplementary Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eD). Additionally, RT-qPCR validation showed a more than 2-fold upregulation in LINC00887 expression within mammospheres compared to its parental cells, while MGC27382 exhibited lower expression levels compared to LINC00887 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH and Supplementary Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eE). In our in-house tissue microarray, LINC00887 is aberrantly overexpressed in BRCA tissues relative to tumor-adjacent control (n\u0026thinsp;=\u0026thinsp;136, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eI). Further analyses revealed that elevated LINC00887 level was positively correlated with tumor size and advanced AJCC stage (Supplementary Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eF and S1G). Strikingly, LINC00887 expression was significantly elevated in CD44⁺/CD24⁻ BCSCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eI and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eJ), suggesting that LINC00887 may contribute to the maintenance of BCSC properties.\u003c/p\u003e\u003cp\u003eConsistently, our \u003cem\u003ein vitro\u003c/em\u003e functional studies revealed that LINC00887 KD dramatically inhibited cell proliferation, reduced the capacity of cellular sphere formation and the proportion of CD44\u003csup\u003e+\u003c/sup\u003eCD24\u003csup\u003e\u0026minus;\u003c/sup\u003e BCSCs, whereas ectopic expression of LINC00887 markedly increased anchorage-dependent growth and anchorage-independent growth capacities (Supplementary Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eA-S2I). As expected, \u003cem\u003ein vivo\u003c/em\u003e limited dilution assay confirmed that LINC00887 KD led to dramatic decreases in both tumorigenic ability and the frequency of BCSCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eK-\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eN and Supplementary Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eJ-S2K). Moreover, to identify biological pathways associated with LINC00887, we conducted GSEA enrichment analysis using TCGA BRCA data (n\u0026thinsp;=\u0026thinsp;1109). Notably, glycolysis signaling pathways are hyper-activated in patients with high LINC00887 expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eO), indicating that LINC00887 may support the maintenance of BCSC properties by modulating glycolysis pathway. To further determine critical metabolic target genes of LINC00887, we conducted screening on glycolytic enzymes (Supplementary Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eL). Among all the genes examined, PKM1 and PKM2 were found to be the most significantly changed by LINC00887 KD (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eP). PKM1 and PKM2 are two alternative splicing isoforms of pyruvate kinase muscle (PKM), serving as rate-limiting enzyme in glycolysis. In various cancer cells, PKM2 predominates as the primary form of PKM to modulate cancer metabolism\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e,\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. We further investigated the clinical relevance of PKM isoforms and found that, compared to normal control, PKM2 expression was significantly elevated in BRCA, whereas PKM1 expression was markedly reduced (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eQ). Additionally, high PKM2 expression was positively correlated with advanced tumor Stage, while low PKM1 expression correlated with higher tumor Stage (Supplementary Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eM and S2N). We also found that PKM2 protein levels were higher in the BRCA CD44\u003csup\u003e+\u003c/sup\u003eCD24\u003csup\u003e\u0026minus;\u003c/sup\u003e subsets, while PKM1 expression remained low (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eQ). Further correlation analysis confirmed a remarkably positive relationship between LINC00887 expression and PKM2 expression, while PKM1 showed a mild negative correlation with LINC00887 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eR), implying that PKM2 and PKM1 are the potential targets of LINC00887. Together, these findings indicate that LINC00887 might activate the glycolytic pathway of BCSCs by regulating PKM1 and PKM2.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eLINC00887 promotes BCSC properties by activating PKM2-mediated glycolysis\u003c/h3\u003e\n\u003cp\u003eThe PKM gene encodes two major isoforms: PKM1 and PKM2. Significant elevation of PKM2 expression was observed in various cancers, whereas the tissue-specific enzyme expressions of PKM1 was mainly expressed in normal cells. It is thought that PKM2 rather than PKM1 promotes glycolysis and tumor progression in cancer. However, the mechanisms driving the shift in PKM isoform expression, particularly in BCSCs, remain poorly understood. Here, we observed a marked increase in PKM2 protein levels and a corresponding decrease in PKM1 in BRCA compared with matched adjacent normal tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eQ). Moreover, we revealed that PKM2, but not PKM1, was highly expressed in CD44\u003csup\u003e+\u003c/sup\u003eCD24\u003csup\u003e\u0026minus;\u003c/sup\u003e BRCA (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eQ). Consistently, Western blot further confirmed the upregulation of PKM2 and the downregulation of PKM1 in BCSCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB), highlighting a potential role for PKM2 in the metabolic reprogramming of BCSCs.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo investigate the regulatory mechanism underlying PKM isoform expression, we focused on the long noncoding RNA. LINC00887 KD significantly increased PKM1 protein levels and decreased PKM2 levels in MCF-MS cells, whereas forced expression of LINC00887 in MCF-7 cells had the opposite effect (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Functionally, LINC00887 KD resulted in a significant reduction in glycolytic rates and lactate levels, while forced expression of LINC00887 significantly increased glycolytic rates and lactate levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD-\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG). Collectively, our data suggest that LINC00887 promotes glycolysis in BCSCs by regulating PKM isoform expression.\u003c/p\u003e\u003cp\u003ePKM two isoforms arise from mutually exclusive alternative splicing of the PKM pre-mRNA, which reflects the inclusion of either exon 9 (PKM1) or exon 10 (PKM2) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH). We analyzed the distribution of these two isoforms of PKM and found that that PKM2, rather than PKM1, predominates in BCSCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eI). To determine whether LINC00887 mediates PKM1/PKM2 splicing, we detected the altered splicing of PKM by semi-quantitative RT-PCR followed by exon10-specific restriction digestion. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eJ, LINC00887 KD notably augmented the splicing of the PKM1 variant while diminishing the PKM2 isoform. Conversely, there was a substantial decrease in PKM1 splicing and an increase in PKM2 splicing in MCF7 cells overexpressing LINC00887 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eJ). Taken together, our results indicate that PKM1 and PKM2 are functionally essential metabolic targets of LINC00887 and contribute to LINC00887 KD-induced glycolytic inhibition in BCSCs.\u003c/p\u003e\n\u003ch3\u003eHnRNPA1 drives PKM alternative splicing to favor PKM2 isoform expression in BCSCs\u003c/h3\u003e\n\u003cp\u003eAlternative splicing regulation involves recognition of cis-acting sequence motifs within pre-mRNA by trans-acting RNA binding proteins (RBPs)\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. PKM1 and PKM2 splice variants result from the recruitment of specific splicing factors to PKM pre-mRNA, thereby driving mutually exclusive alternative splicing events. To identify potential splicing factors involved in the alternative splicing of PKM pre-mRNA in BCSCs, we compared the expression of splicing factors in MCF-7 MS vs MCF-7 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). hnRNPA1 and QKI were two of the most highly expressed splicing factors in MCF-7 MS cells relative to MCF-7 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB and Supplementary Fig. S3A). MOE docking analysis revealed that both hnRNPA1 and QKI bind to PKM pre-mRNA, with hnRNPA1 exhibiting a substantially higher binding affinity (S = \u0026minus;\u0026thinsp;63.06) than QKI (S = \u0026minus;\u0026thinsp;44.07) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC-\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD and Supplementary Fig. S3B). Further structural analysis revealed that hnRNPA1 recognizes the UAGGG motif located within the flanking region of exon 9 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). Subsequent RNA pull-down assays using biotin-labeled PKM probes confirmed the direct interaction of wild-type (WT) PKM, but not mutant (MUT) PKM, with the hnRNPA1 protein (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). Furthermore, forced expression of hnRNPA1 promotes trans-expression of PKM1 to PKM2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG). Similar patterns were observed by western blotting in hnRNPA1 overexpressing MCF7 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eH and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eI). To evaluate the clinical relevance of the hnRNPA1/PKM2 axis, we checked the correlation between hnRNPA1 and PKM2 expression in BRCA patient cohorts. HnRNPA1 expression showed a positive correlation with PKM2 expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eJ), supporting the regulation of PKM2 by hnRNPA1. GSEA analysis of TCGA BRCA data revealed that glycolysis-related gene signatures were significantly enriched in hnRNPA1⁺ samples (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eK). As expected, forced expression of hnRNPA1 significantly enhanced glycolytic rates (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eL).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eFurthermore, we investigated the clinical relevance of hnRNPA1 protein and found that compared to normal control, hnRNPA1 protein was highly expressed in BRCA (Supplementary Fig. S3C-S3E). Moreover, hnRNPA1 expression was elevated in CD44\u003csup\u003e+\u003c/sup\u003eCD24\u003csup\u003e\u0026minus;\u003c/sup\u003e BRCA than in non-CD44\u003csup\u003e+\u003c/sup\u003eCD24\u003csup\u003e\u0026minus;\u003c/sup\u003e BRCA (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eM). Consistently, western blot assays revealed that the expression of hnRNPA1 was markedly upregulated in BCSCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eN). KM Plotter analysis showed that high hnRNPA1 protein expression was associated with poor overall survival in BRCA patients (n\u0026thinsp;=\u0026thinsp;108, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Supplementary Fig. S3F). Further analysis revealed that elevated hnRNPA1 expression was positively associated with adverse clinical features, including advanced stage (Stage III, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), size\u0026thinsp;\u0026gt;\u0026thinsp;2.5cm (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), and lymphatic metastasis (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eO and Supplementary Fig. S3G-S3I). Collectively, these findings demonstrate that hnRNPA1 directly binds PKM pre-mRNA and promotes mutually exclusive splicing favoring PKM2 production, thereby enhancing glycolysis in BRCA.\u003c/p\u003e\n\u003ch3\u003eLINC00887 directly binds to hnRNPA1 to mediate its expression and sustain BSCSs properties\u003c/h3\u003e\n\u003cp\u003eGiven that both LINC00887 and hnRNPA1 are involved in mutually exclusive splicing of PKM pre-mRNA, we investigated whether LINC00887 promotes PKM2 isoform expression via hnRNPA1. Linear regression analysis revealed a significant positive correlation between LINC00887 and hnRNPA1 protein expression in BRCA (n\u0026thinsp;=\u0026thinsp;136, r\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.34, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). To further elucidate the underlying interaction of hnRNPA1 in the context of LINC00887 mediated BCSCs properties, we employed tartaglialab (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.tartaglialab.com/\u003c/span\u003e\u003cspan address=\"http://www.tartaglialab.com/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) to evaluate the binding propensity between LINC00887 nucleotide sequences and hnRNPA1 amino acid residues. LINC00887 (2000 nt-2500 nt) exhibited strong interaction signal with hnRNPA1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). MOE docking analysis predicted that LINC00887 could bind to the lysine residues 144\u0026ndash;145 in the RRM domain of hnRNPA1(S=-66) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Additionally, we conducted RIP assays and confirmed that hnRNPA1 protein strongly binded with LINC00887 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). Interestingly, LINC00887 KD did not alter the mRNA level of hnRNPA1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE) but significantly reduced its protein levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF), suggesting that LINC00887 may regulate hnRNPA1 at the post-translational level. Conversely, hnRNPA1 overexpression did not alter LINC00887 expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG). Collectively, our data suggest that hnRNPA1 is a downstream target of LINC00887.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo determine the roles of hnRNPA1 in LINC00887-mediated cell proliferation and BCSCs self-renewal, we ectopically expressed hnRNPA1 in LINC00887 KD MS (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH) to conduct rescue assays. As expected, forced expression of hnRNPA1 largely rescued the inhibitory effects of LINC00887 KD on cell proliferation/growth in MCF7 MS and T47D MS (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eI). Moreover, forced expression of hnRNPA1 also significantly rescued LINC00887 KD-induced inhibitory effect on the biological characteristics of BCSCs, including the percentage of CD44\u003csup\u003e+\u003c/sup\u003eCD24\u003csup\u003e\u0026minus;\u003c/sup\u003e populations, and the self-renewal abilities in both MCF-7 MS and T47D MS cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eJ and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eK). Taken together, our data indicate that hnRNPA1 are bona fide and functionally essential targets of LINC00887 and are responsible for LINC00887 KD-induced inhibitory effects on self-renewal/repopulation capacity in BCSCs.\u003c/p\u003e\n\u003ch3\u003eInhibition of hnRNPA1 ubiquitination by LINC00887 trigger PKM2 splicing and glycolysis Activation\u003c/h3\u003e\n\u003cp\u003eOur data showed that hnRNPA1 is required for LINC00887-driven BCSC maintenance. Next, we sought to elucidate the mechanism by which LINC00887 regulates hnRNPA1 protein levels in BCSCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Our results showed that LINC00887 KD significantly decreased hnRNPA1 protein expression, without affecting its mRNA levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF), implying that LINC00887 likely modulates hnRNPA1 through a post-translational mechanism. Accumulating evidence suggests that the protein stability of hnRNPA1 is precisely controlled by ubiquitin-proteasome system (UPS)\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e,\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e,\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. We further identified potential ubiquitination sites on hnRNPA1, specifically at lysine residues 105, 144, 145, 179, and 183, using the BDM-PUB database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://bdmpub.biocuckoo.org/\u003c/span\u003e\u003cspan address=\"http://bdmpub.biocuckoo.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). MOE Docking analysis predicted a binding interaction between LINC00887 and hnRNPA1 protein (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). RNA pull-down assays further confirmed that the WT 887 probe, but not the Mut 887 probe, significantly pulled down endogenous nuclear hnRNPA1 protein, which enrichment was notably diminished following the knockdown of LINC00887 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). Strikingly, LINC00887 binds to the lysine 144\u0026ndash;145 site of hnRNPA1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC), which corresponds to its predicted ubiquitination sites. Functionally, overexpression of LINC00887 led to a noticeable decrease in the ubiquitination of endogenous hnRNPA1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). Moreover, cycloheximide (CHX) chase assays demonstrated that LINC00887 overexpression significantly prolonged the half-life of hnRNPA1 protein in MCF-7 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF), supporting its role in stabilizing hnRNPA1 by suppressing its degradation. Taken together, our data suggest that LINC00887 directly binds with hnRNPA1, inhibits its ubiquitination and in turn stabilizes hnRNPA1 in BRCA.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo determine the role of hnRNPA1 in LINC00887 KD-induced glycolytic inhibition in BCSCs, we conducted rescue assays by overexpressing hnRNPA1 in LINC00887-depleted cells. First, we detected the expression of PKM2 and PKM1 after co-transfecting MCF-7 MS and T47D MS cells with LINC00887 silencing and hnRNPA1 overexpression lentivirus. Western blot assays confirmed that there was a significant downregulation of PKM2 expression and an upregulation of PKM1 expression in the sh887 group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG). Notably, overexpression of hnRNPA1 could reverse these changes in protein expression induced by LINC00887 KD (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG and Supplementary Fig. S4A), indicating that LINC00887 mediates the expression of PKM1 and PKM2 in a hnRNPA1-dependent manner. Moreover, we observed that forced expression of hnRNPA1 could rescue LINC00887 KD-induced reduction of CD44 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG), demonstrating that hnRNPA1 is essential for the LINC00887-mediated self-renewal of BCSCs. Further analysis revealed that overexpression of hnRNPA1 rescued the reduced lactate accumulation induced by silencing LINC00887 in BCSCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eH). Strikingly, overexpressing hnRNPA1 also restored the glycolytic capacities impaired by LINC00887 KD in MCF-7 MS (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eI and Supplementary Fig. S4B). These results strongly confirmed that the LINC00887/hnRNPA1/PKM2 axis activates glycolysis and contributes to the maintenance of BCSCs stemness.\u003c/p\u003e\u003cp\u003e\u003cb\u003eSilencing LINC00887 reduces stemness and the growth of BCSCs xenografts via inhibiting hnRNPA1 splicing to form PKM2 formation\u003c/b\u003e\u003c/p\u003e\u003cp\u003eFinally, we used cell-line-derived xenograft (CDX) models to evaluate the effect of LINC00887-hnRNPA1-PKM2 axis on BRCA progression \u003cem\u003ein vivo\u003c/em\u003e. We stably expressed hnRNPA1 in sh887 and shCtrl MCF-7 MS, injected 3x10^5 cells into 6-week-old BALB/c nude female mice and monitored BRCA progression via bioluminescent imaging (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). As expected, LINC00887 KD significantly reduced the bioluminescence signals, dramatically inhibited tumor growth, and substantially prolonged the overall survival in the recipient mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB-\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eG). Forced expression of hnRNPA1 could totally reverse the LINC00887 KD-induced phenotypes \u003cem\u003ein vivo\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB-\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eG), demonstrating that the inhibitory effect of LINC00887 KD on BRCA progression \u003cem\u003ein vivo\u003c/em\u003e could be largely due to the dysregulation of hnRNPA1.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eIn addition, immunohistochemistry revealed that LINC00887 KD markedly downregulated the protein levels of PKM2, while significantly increasing the protein levels of PKM1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eH and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eI). Meanwhile, we further confirmed that forced expression of hnRNPA1 could rescue LINC00887 KD-induced reduction of PKM2 and CD44, and the increase of PKM1 by western blot (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eJ and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eK). Also, we found that LINC00887 KD showed consistently significant inhibitory effects on glycolytic rates \u003cem\u003ein vivo\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eL). More strikingly, glycolytic inhibition caused by LINC00887 KD could be largely rescued by overexpression of hnRNPA1 \u003cem\u003ein vivo\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eL). Collectively, our data indicate that hnRNPA1 and PKM2 are functionally essential downstream targets of LINC00887 and are responsible for its oncogenic role in BRCA progression.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIt is well established that BCSCs play critical roles in BRCA progression and recurrence. Understanding the molecular determinants of BCSCs will help develop targeted strategies and improve clinical outcomes in BRCA patients. Glycolysis reprogramming is an emerging hallmark of BRCA, contributing to the maintenance of tumorigenic potential. Recent advancements have disclosed this metabolic phenotypic difference between cancer cells and their normal counterparts, offering promising opportunities to target key metabolic enzymes in cancer cells\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e,\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. Here, we revealed that BCSCs exhibit enhanced glycolysis, which is strongly dependent on PKM2. Moreover, we reported that lncRNA heterogeneity contributes to metabolic reprogramming in BRCA, wherein LINC00887 regulates key metabolic enzymes such as PKM1 and PKM2. Furthermore, we demonstrate that LINC00887 blocks the ubiquitination of hnRNPA1 and subsequently switches PKM1 to PKM2 and enhances aerobic glycolysis, which supports the self-renewal and survival of BCSCs.\u003c/p\u003e\u003cp\u003eThrough transcriptome-wide microarray and Seahorse Glycolytic Rate Assay, we revealed that BSCSs are characterized by their enhanced glycolytic activity, a feature that distinguishes them from non-stem cancer cells and highlights their metabolic plasticity. Emerging data have suggested that the dysregulation of the expression and activities of key central metabolic enzymes contribute to metabolic reprogramming in cancer. PKMs serve as a crucial rate-limiting step in glycolysis, determining the fate of pyruvate and the flow of carbon through this central metabolic pathway. PKM exists in two isoforms, PKM1 and PKM2, which arise from the alternative splicing of exons 9 and 10 of the PKM gene, respectively\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. While PKM1 is prevalent in most differentiated tissues, PKM2 is predominantly expressed in proliferating tissues and tumors, where it facilitates glycolytic flux and anabolic growth. Recent works on PKM in cancers revealed that switching PKM2 to PKM1 reverses aerobic glycolysis to oxidative phosphorylation and delays tumorigenesis, suggesting PKM2 as an appealing target for cancer therapy \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Here, we revealed that PKM2 is significantly upregulated in BRCA particularly in CD44⁺CD24\u003csup\u003e\u0026minus;\u003c/sup\u003e BCSCs, whereas PKM1 expression remains low. Importantly, KD of either LINC00887 or hnRNAP1 induces a splicing switch from PKM2 to PKM1 in BCSCs, triggering a potential therapeutic strategy to selectively suppress PKM2-driven glycolysis in BRCA.\u003c/p\u003e\u003cp\u003ehnRNPA1 could serve as an oncofetal gene that is highly upregulated in a various types of cancer, such as pancreatic cancer\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e,\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e, gastric cancer\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e and chronic myelogenous leukemia\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. Here, we showed that hnRNPA1 is significantly overexpressed in BCSCs than non-BCSCs. Our functional study revealed that hnRNPA1 is required for the maintenance and self-renewal of BCSCs, clarifying the function of hnRNPA1 as oncogenic in BRCA. Previous studies showed that hnRNPA1 acts as a splicing factor, mediating PKM splicing. Consistently, we found that hnRNPA1 binds to the flanking sequences of PKM pre-mRNA exon 9, facilitating the inclusion of exon 10 and the production of PKM2 isoform in BCSCs. This regulation underscores the intricate control mechanisms that govern isoform switching and metabolic adaptation in cancer cells. By interacting with PKM pre-mRNA, hnRNPA1 ensures a preferential splicing outcome that aligns with the metabolic needs of BCSCs. This splicing regulation is critical for maintaining the glycolytic flux and the anabolic processes essential for BCSCs' self-renewal and proliferation.\u003c/p\u003e\u003cp\u003eLINC00887 has been investigated in several malignancies, with seemingly contradictory findings regarding its function. Tous et al. revealed that LINC00887 was highly expressed in thyroid Cancer and promoted cell proliferation and invasion\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. However, LINC00887 has also been reported to be serving as a tumor suppressor in ovarian high-grade serous carcinoma\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e, cervical cancer\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e, and lung cancer\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. Our study has unveiled an upregulation of LINC00887 levels in BRCA compared to their counterparts. Further analysis revealed that LINC00887 is expressed at a significantly higher level in BCSCs than in non-BCSCs. We also showed that KD of LINC00887 inhibited cell proliferation and induced apoptosis in BRCA cells. Furthermore, our in vitro and in vivo functional studies revealed that LINC00887 maintains the self-renewal of BCSCs by promoting PKM2 expression and reprogramming glucose metabolism. hnRNPA1 can be post-translationally modified by ubiquitination, which mediates its stability\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e,\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e,\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e,\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. In this study, we identified that LINC00887 upregulates PKM2 by binding to hnRNPA1, thereby concealing its ubiquitination site, preventing its degradation, and ensuring its stability. The stabilization of hnRNPA1 by LINC00887 underscores a layer of post-translational regulation that fine-tunes the expression of metabolic enzymes in response to cellular and environmental cues.\u003c/p\u003e\u003cp\u003eIn summary, our study demonstrated the strong dependency of LINC00887 for the self-renewal of BCSCs. We also revealed a previously unappreciated signaling involving LINC00887, ubiquitination, hnRNPA1, and PKM splicing, which controls aerobic glycolysis and exerts its critical role in the pathogenesis of BRCA, cancer metabolism, and BSCSs self-renewal. Considering the essential roles of LINC00887 in BRCA pathogenesis and the maintenance of BCSCs, targeting LINC00887 LINC00887/hnRNPA1/PKM2 signaling holds great therapeutic potential for the treatment of BRCA patients by eliminating BCSCs. Our work provides novel insights into the combination of epigenetic-modulating agents and cancer metabolism-targeting agents for cancer therapy.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003eClinical samples\u003c/h2\u003e\u003cp\u003eIn the current study, we included 136 paired BRCA tissues and matched adjacent-tumor controls obtained from the Department of General Surgery of the First Hospital of China Medical University and the Department of Medical Oncology of Cancer Hospital of China Medical University between March 2013 and December 2017. This study was approved by the Medical Ethics Committee of China Medical University, and all patients signed written informed consent forms before enrollment. The tissues were immediately snap-frozen in liquid nitrogen after separation and stored at -80\u0026deg;C until use. Information on clinicopathological parameters can be found in Table\u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eMicroarray analysis\u003c/h2\u003e\u003cp\u003eTotal RNA was extracted from cells using TRIzol reagent according to the manufacturer\u0026rsquo;s instructions (15596026, Invitrogen). RNA samples were subjected to GeneChip\u0026reg; Human Transcriptome Array 2.0 (HTA2.0, Affymetrix, USA), which includes global profiling of full-length transcripts. The cDNA labeling, microarray hybridization and data acquisition were performed according to the manufacturer\u0026rsquo;s instructions. Feature Extraction software (version10.7.1.1, Agilent Technologies) was used to extract raw data from microarray images. Genespring software (version 13.1; Agilent Technologies) was employed for quantile normalization and background correction. Gene Set Enrichment Analysis (GSEA) with differentially expressed genes was performed to identify enriched pathway. Differentially expressed genes between BCSCs and non-BCSCs were identified with Log\u003csub\u003e2\u003c/sub\u003e Fold Change\u0026thinsp;\u0026ge;\u0026thinsp;1.0 and P\u0026thinsp;\u0026le;\u0026thinsp;0.05. The raw data have been deposited in NCBI\u0026rsquo;s Gene Expression Omnibus (GEO) under accession number GSE273726.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eCell culture\u003c/h2\u003e\u003cp\u003eHuman breast cancer cell lines including MCF-7 (ATCC cat.HTB-22, RRID: CVCL_0031) and T47D (ATCC cat.HTB-133, RRID: CVCL_0553) were obtained from the American Type Culture Collection (ATCC). T47D were cultured in DMEM supplemented with 10% FBS; MCF7 was maintained in DMEM supplemented with 10% FBS and 0.01 mg/ml insulin (I8830, Solarbio). Mammospheres were cultured as reported previously by He et al \u003csup\u003e44\u003c/sup\u003e. Briefly, MCF7 sphere (MCF7 MS) and T47D sphere (T47D MS) were cultured in suspension in serum-free DMEM-F12 supplemented with 2% B27 (17504044, Invitrogen), 20 ng/ml human recombinant EGF (AF-100-15, Peprotech), 10 ng/ml human recombinant basic FGF (100-18B, Peprotech). Cells grew under these conditions as nonadherent spheroid cell masses, named mammospheres (MS). Penicillin-Streptomycin (15140122, Gibco) and Plasmocin prophylactic (ant-mpp, InvivoGEN) were supplemented to all the media to prevent potential contamination.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eLentivirus infection\u003c/h2\u003e\u003cp\u003eLentiviral particles for shCtrl, sh887#1, sh887#2, GV146-LINC00887 and PCDH-hnRNPA1 were purchased from Shanghai Genechem Co., Ltd. Sh887 sequence is seen in Supplementary Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e. On the first day, 2\u0026times;10^5 cells were seeded in 6-well plates. The following day, lentivirus (shCtrl,MOI\u0026thinsp;=\u0026thinsp;10; sh887, MOI\u0026thinsp;=\u0026thinsp;20) was directly added to the cells in the presence of 5 \u0026micro;g/ml polybrene (H9268, Sigma-Aldrich). Lentivirus were incubated with the cells for 24 hours, after which the medium was replaced with fresh medium. 48 hours after infection, the infected cells were subjected to puromycin (2\u0026micro;g/ml) select stably transfected cell lines (P8833, Sigma-Aldrich). This screening procedure was repeated 3\u0026ndash;5 times until all cells expressed green fluorescent protein.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eRT-PCR assay and qPCR assay\u003c/h2\u003e\u003cp\u003eTotal RNA was extracted from cells using TRIzol reagent according to the manufacturer\u0026rsquo;s instructions (15596026, Invitrogen). Reverse transcriptase reaction was performed with 500\u0026ndash;1000 ng of total RNA in a total reaction volume of 20 \u0026micro;L using HiScript III RT SuperMix for qPCR (+\u0026thinsp;gDNA wiper) (R323-01, Vazyme). Quantitative real-time PCR (qPCR) was performed with ChamQ SYBR qPCR Master Mix (Low ROX Premixed) (Q331-02, Vazyme) on the QuantStudio 7 Flex PCR system (Applied Biosystems). Target gene expression levels were normalized to β-actin. Each reaction was performed three times, and relative mRNA abundance was calculated using the contrast ΔΔCT method. The calculated mRNA abundance of glycolytic enzyme genes was further transformed by Z-score normalization to create a heat map. All primers used for qPCR analysis were ordered from Sangon Biotech (Shanghai, China). The primers used for qPCR analysis were listed in Table S3.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eProtein extraction and Western blot analysis\u003c/h2\u003e\u003cp\u003eFor protein extraction, cells were collected and washed twice with chilled PBS. Total proteins were extracted with RIPA buffer (P0013B, beyotime) containing protease inhibitor and phosphatase inhibitor cocktail (P1050, beyotime). The separation of nuclear and cytoplasmic fractions was performed using the PARIS\u0026trade; Kit (Thermo Fisher) according to the manufacturer's instructions. Protein concentration was determined by BCA Protein Assay Kit (P0012, beyotime). Protein lysates were diluted with 5X SDS-PAGE Sample Loading Buffer (P0015L, beyotime) and denatured at 99℃ for 10min.\u003c/p\u003e\u003cp\u003eFor western blot, equal amounts of proteins were separated by 10% SDS-PAGE gels and transferred to polyvinylidene fluoride (PVDF) membranes (IPVH00010, Millipore). Then, the membranes were blocked with 5% milk and incubated with specific primary antibodies overnight at 4℃. Finally, membranes were incubated with secondary antibody and visualized by immunoblotting with Pierce ECL Western blotting Substrate (32106, Thermo Fisher). Primary antibodies used in this study were as follows: PKM1 (1:3000; 15821-1-AP, Proteintech), PKM2 (1:3000; 15822-1-AP, Proteintech), hnRNPA1 (1:2000; Cat. # 8443, Cell signaling technology), CD44 (1:1000; ab157107, Abcam), PARA (1:1000, Cat. # 9542T, Cell signaling technology), β-actin (1:5000; 66009-1-Ig, Proteintech) and GAPDH (1:5000, 60004-1-Ig, Proteintech).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eTissue microarray (TMA) and immunohistochemistry (IHC)\u003c/h2\u003e\u003cp\u003eTMA and IHC assays were performed as previously described \u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. Briefly, BRCA tissue and matched tumor-adjacent control samples in paraffin blocks were visualized using hematoxylin and eosin-stained slides. Tissue cores with a diameter of 1.5 mm were extracted from each paraffin block and precisely arranged into a new paraffin block containing multiple cores using tissue microarray technology (Pathology Equipment). Each TMA section (4 \u0026micro;m) was deparaffinized in xylene and rehydrated in a graded ethanol series. The sections were then incubated overnight at 4\u0026deg;C with specific primary antibodies against PKM1(15821-1-AP,1:200, Proteintech, USA), PKM2(15822-1-AP,1:200, Proteintech, USA), hnRNPA1(15821-1-AP,1:1000, Proteintech, USA),CD44((960-MSM1-P0, 1:100; Thermo Fisher Scientific, USA), CD24(MA5-11828,1:200, Thermo Fisher Scientific, USA).The sections were subsequently incubated with a biotinylated secondary antibody at 37\u0026deg;C for 30 minutes. Horseradish peroxidase-coupled streptavidin (Dako, Glostrup) was then added and incubated for 30 minutes, followed by DAB (3,3-diaminobenzidine) staining. Finally, the sections were counterstained with hematoxylin and mounted with neutral resin. Protein expression levels were observed and counted under a microscope (Eclipse 8i, Nikon). Immunoreactivity was evaluated independently by two investigators according to the percentage of stained cells and staining intensity. Staining intensity was categorized as 0 (absent), 1 (weak), 2 (moderate), or 3 (strong). The final score was calculated by multiplying the intensity score by the percentage score of positively stained cells, yielding a total range of 0\u0026ndash;300. To establish an expression-level cutoff (low vs. high), receiver operating characteristic (ROC) curve analysis was applied, with the optimal cutoff determined by maximizing the area under the curve (AUC) and minimizing the sum of sensitivity and 1-specificity for clinical variables.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003eLncRNA in situ hybridization (ISH) assay\u003c/h2\u003e\u003cp\u003eRNA ISH was performed with Enhanced Sensitive ISH Detection Kit Ⅰ (POD) (MK1030, Boster) according to the manufacturer\u0026rsquo;s instructions. After deparaffinization and deproteinization, the sections were pre-hybridized in pre-hybridization solution at 42\u0026deg;C for 2 hours. The sections were then incubated with dig-labeled probe solution at 37\u0026deg;C overnight. Following stringent washing, the slides were exposed to a streptavidin-peroxidase reaction system and stained with DAB (ZLI-9017, Zsbio) for 2 minutes. Subsequently, 0.1% hematoxylin (H8070, Solarbio) was used to counterstain the slides for 5 minutes. LINC00887 expression levels were observed and counted under a microscope (Nikon, Tokyo, Japan). The expression score for LINC00887 was calculated as described above of ICH. The LINC00887 probe is shown in Table S4.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003eCell proliferation assay\u003c/h2\u003e\u003cp\u003eCell proliferation was assessed by Cell Counting Kit-8 (CCK-8) assay (341\u0026ndash;07761, Dojindo) following the manufacturer\u0026rsquo;s instructions. Briefly, cells infected with indicated lentivirus were seeded on 96-well plates in triplicates at a density of 3000 cells/well in a final volume of 100 ml. Then, 10 \u0026micro;l CCK-8 solution was added to each well at indicated time points and incubated at 37\u0026deg;C for 2\u0026ndash;4 h. Finally, the absorbance at 450 nm was determined using an Anthos 2010 microplate reader (Anthos Labtec Instruments GmbH, Austria).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003eSemi-quantitative RT-PCR and PKM splicing assays\u003c/h2\u003e\u003cp\u003ePKM splicing assays were performed as previously described\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Briefly, total RNA was extracted from cells using TRIzol reagent. Semi-quantitative RT-PCR were used to analyze alternative spliced products. The PCR products of PKM were digested with FastDigest PstI endonuclease (Thermo, Scientific). Finally, the digested products were resolved by 6% non-denaturing PAGE and images were taken by BioSpectrum AC Imaging System (UVP). The primers for PKM variants are shown in Table S3.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003eIn vivo limiting dilution assay\u003c/h2\u003e\u003cp\u003eFor in vivo limiting dilution assay, 3 x 10\u003csup\u003e4\u003c/sup\u003e, 3 x 10\u003csup\u003e5\u003c/sup\u003e, 3 x 10\u003csup\u003e6\u003c/sup\u003e cells control or LINC00887 KD MCF7 MS were injected subcutaneously into BALB/c nude mice. The number of recipient mice developed tumor within 20 days post-transplantation was counted for each group with each dose of donor cells. The frequency of BCSCs were estimated by ELDA software (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://bioinf.wehi.edu.au/software/elda/\u003c/span\u003e\u003cspan address=\"http://bioinf.wehi.edu.au/software/elda/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\u003ch2\u003eSoft-agar colony formation assay\u003c/h2\u003e\u003cp\u003eSoft agar colony formation assay was carried out as described previously\u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. Briefly, 1.2% agarose gel (50004, Lonza Rockland) was mixed with phenol red-free DMEM medium supplemented with 20% FBS and 2% penicillin-streptomycin in 6-well plates as the bottom layer. Mix the cells (10\u003csup\u003e3\u003c/sup\u003e cells/ml) into 0.6% agarose gel and phenol red‐free DMEM medium (1:1) supplemented with 20% FBS and 2% penicillin-streptomycin as the top layer. An additional 600 \u0026micro;l medium were added to the uppermost layer, and the medium was added every 3\u0026ndash;4 days. After 2\u0026ndash;3 weeks of incubation at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e, the colonies larger than 150 \u0026micro;m in diameter were counted with MTT (5mg/ml) staining.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\u003ch2\u003eSpheroid formation assay\u003c/h2\u003e\u003cp\u003eMammospheres cells (2000cells/ml) were collected and re-suspended in DMEM/F-12 supplemented with 2% B27 (17504044, Invitrogen), 20 ng/ml human recombinant EGF (AF-100-15, Peprotech), 10 ng/ml human recombinant basic FGF (100-18B, Peprotech) in 6-well Ultra-Low Attachment Microplates plates (3473, Corning\u0026trade;). 0.5 mL of fresh medium was added to each well every 3 days. After 2 weeks of incubation at 37\u0026deg;C in a humidified environment with 5% CO\u003csub\u003e2\u003c/sub\u003e, the number of spheres\u0026thinsp;\u0026gt;\u0026thinsp;100 \u0026micro;m in diameter was counted under an inverted microscope (Nikon TE2000-U, Japan).\u003c/p\u003e\u003cdiv id=\"Sec23\" class=\"Section3\"\u003e\u003ch2\u003eFlow cytometry assays\u003c/h2\u003e\u003cp\u003eThe cells were harvested, washed with chilled PBS 3 times, resuspended in 100ul Flow Cytometry staining buffer (00-4222-26, eBioscience) containing APC-conjugated CD44 antibody (103011, BioLegend) and PE-conjugated CD24 antibody (311105, BioLegend) and incubated at 4\u0026deg;C for 30 min in the dark. After incubation, the cells were washed with PBS 3 times. Then, single-cell suspensions were subjected to analysis by MACSQuatTM Flow cytometer (Becton-Dickinson, USA) within 2 h.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec24\" class=\"Section2\"\u003e\u003ch2\u003eRNA pull-down assay\u003c/h2\u003e\u003cp\u003eRNA pull-down was performed with Pierce\u0026trade; Magnetic RNA Protein Pull Down Kit (20164, Thermo Fisher Scientific) following the manufacturer\u0026rsquo;s instructions. Briefly, biotin-labeled probes were conjugated to Streptavidin agarose resin beads (Thermo Fisher Scientific). Then, biotin-labeled probe-conjugated streptavidin beads were incubated with nuclear extracts in Protein-RNA binding buffer overnight at 4\u0026deg;C. After washed 4 times with 1\u0026times; washing buffer, the protein-RNA-beads mixture was dissolved in 1\u0026times; SDS buffer and analyzed by western blot. The LNC887 and PKM probes are shown in Table S5.\u003c/p\u003e\u003cdiv id=\"Sec25\" class=\"Section3\"\u003e\u003ch2\u003eRNA Immunoprecipitation and Semi-quantitative\u003c/h2\u003e\u003cp\u003eRIP was performed as described previously with some modifications\u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. Briefly, cells were collected, washed with chilled PBS, and lysed with 1ml Co-IP lysis buffer (PR20037, Proteintech) with protease inhibitor and RNase inhibitor. The lysates were centrifuged at 12,000 g for 10 min at 4\u0026deg;C. Then, supernatants were collected. 5% of lysate was saved as input. HnRNPA1 antibody (11176-1-AP, Proteintech) and IgG antibody (30000-0-AP, Proteintech) were conjugated to Protein A/G Magnetic Beads by rotation at 4\u0026deg;C for 4 h. Supernatants were incubated with the antibody-conjugated beads with rotation at 4\u0026deg;C overnight. Afterward, beads were washed 3 times with chilled RIP buffer and followed by Proteinase K (E00492, Thermo Fisher Scientific) treatment at 55\u0026deg;C for 1h. The input and co-immunoprecipitated RNA were extracted with TRIzol reagent and analyzed by Semi-quantitative RT-PCR.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec26\" class=\"Section3\"\u003e\u003ch2\u003eLactate Levels\u003c/h2\u003e\u003cp\u003eLactate levels were determined with CheKine\u0026trade; Micro Lactate Assay Kit (KTB1100, Abbkine) following the manufacturer\u0026rsquo;s instructions. Briefly, the cell supernatant was harvest, filtered with 5 kDa MWCO Amicon\u0026reg; Ultra Centrifugal Filter (UFC901008, Millipore) for deproteinization. The deproteinized samples were then added to a 96-well plate in triplicates with appropriate dilution and incubated with the lactate reaction mix at 37\u0026deg;C for 30 minutes in the dark. The absorbance was measured at a wavelength of 450 nm and the lactate levels were calculated with a standard curve.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec27\" class=\"Section3\"\u003e\u003ch2\u003eMeasurements of ECAR Using the XFe24 extracellular flux analyzer\u003c/h2\u003e\u003cp\u003eECAR was determined with a Seahorse XFe24 Extracellular Flux Analyzer and a Seahorse XF Glycolytic Rate Assay Kit (102342-100, Agilent Technologies) according to the manufacturer\u0026rsquo;s instructions. In brief, 1.5 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells per well were seed in XF24 cell plates. Cells were cultured in XF RPMI medium (without phenol red) supplemented with 2 mM glutamine, 10 mM glucose, 1 mM pyruvate, and 5 mM HEPES in a CO2-free incubator for 1h prior to the assay. The glycolytic rates were analyzed using a Seahorse Bioscience extracellular flux analyzer (Seahorse XF24 FluxPak, Agilent Technologies).\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec28\" class=\"Section2\"\u003e\u003ch2\u003eCycloheximide (CHX) treatments\u003c/h2\u003e\u003cp\u003eAfter MCF-7 cells were transfected with the overexpressed 887 plasmid, cells were treated with CHX (HY-12,320, MCE), and then the protein was collected at 0h,3h and 5h, respectively WB analysis was performed.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec29\" class=\"Section2\"\u003e\u003ch2\u003eXenograft model\u003c/h2\u003e\u003cp\u003eFour-week-old female BALB/c-nu mice were purchased from the Hua Fukang Biological Technologies Inc. The mice were acclimated to the new environment for one week before the experiment. Prior to tumor cell inoculation, they were randomly divided into groups with approximately equal numbers. The researcher conducting the experiment was blinded to the grouping of the animals.\u003c/p\u003e\u003cp\u003eA total of 3 x 10^6 MCF7 MS were injected subcutaneously into the right armpit region of each mouse. All protocols followed the Regulations of Experimental Animal Administration issued by the Ministry of Science and Technology of the People's Republic of China. Once the tumors became visible, their weight and size were measured every five days. Body weight and tumor growth were also assessed every five days. Tumor number or tumor weight in each group was assessed in a blinded manner.\u003c/p\u003e\u003cp\u003eTumor volume was calculated using the equation V\u0026thinsp;=\u0026thinsp;0.5 \u0026times; D \u0026times; d^2, where V represents the tumor volume, D is the longitudinal diameter, and d is the latitudinal diameter. Thirty days after injection, the mice were sacrificed, and the subcutaneous tumors were isolated and measured. Additionally, tumor tissue was fixed in 10% formalin for further experiments.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec30\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eAll statistical analyses were performed using the SPSS 26.0 software package (SPSS Inc., Chicago, USA) and GraphPad Prism 8 software (GraphPad, USA). A P-value of less than 0.05 was considered statistically significant. Pearson's χ2 or Fisher's exact test was employed to determine the associations between indicator expression and clinicopathological parameters in BRCA patients. Linear regression analysis was used to analyze the correlation of LINC00887 with other indicators. Student's t-test (two-tailed), Wilcoxon's test, or Welch's t-test were used to compare significant differences between groups for paired and unpaired continuous variables, presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (s.d.) or median (quartile). The Kaplan-Meier method and Log-rank test were used to estimate prognosis.\u003c/p\u003e\u003cp\u003eFor cell experiments, the sample size was determined to be adequate based on the magnitude and consistency of measurable differences between groups, typically three or more samples. For the xenograft mouse experiments, no statistical method was used to predetermine the sample size, which was based on previous experimental observations. The sample size for each experiment is shown in the figure legend. No data were excluded from the analysis.\u003c/p\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analyzed in this article were included within the article and the additional files. Please contact the corresponding author for data requests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eACKNOWLEDGEMENTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by National Natural Science Foundation of China (NSFC, No.82473134, No.81902708, No.82400216, China), Liaoning Province Higher Education Scientific Research Funding Project (No.JYTM20230128, No.2023JH2/2020008, China), Science and technology innovation team project of China Medical University (No.CXTD2022007, China), \u0026quot;Xingliao Talent Program\u0026quot; of Liaoning Province (XLYC2403200, China). Liaoning Province Science and Technology Plan Project - Special Project on Technological Innovation Facilitating the High-Quality Development of China Medical University( 2025JH2/102800061).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAUTHOR CONTRIBUTIONS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eX. Lv: Conceptualization, resources, formal analysis, supervision, validation, investigation and writing\u0026ndash;original draft. L. Han: Conceptualization, resources, funding acquisition, methodology and writing\u0026ndash;original draft. W. Tong: Conceptualization, resources, formal analysis, supervision. X. Sun: Methodology. Y. Yan: Methodology, funding acquisition. S. Zhou: Methodology, validation. S. Xu: Methodology, validation. D. Zhang: Methodology, validation. J. Wang: Methodology, validation. J. Liu: Methodology, validation. Y. Zhang: Methodology, validation. H. Zhao: Methodology. W. Yao: Methodology. M. Wei: Conceptualization, data curation, supervision, funding acquisition, project administration, writing\u0026ndash;review and editing. B. Chen: Conceptualization, resources, methodology, and project administration. M. He: Conceptualization, data curation, supervision, funding acquisition, project administration, writing\u0026ndash;review and editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eETHICS APPROVAL AND CONSENT TO PARTICIPATE\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal experiments were performed in accordance with the relevant institutional and national guidelines and the regulations of the Medical Laboratory Animal Health Committee of China Medical University (CMU20241515). This study was approved by the Clinical Research Ethics Committee of China Medical University (No. 2019080).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCONSENT FOR PUBLICATION\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCOMPETING INTERESTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCONSENT FOR PUBLICATION\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll of the authors have written informed consent.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eMartelotto, L. G., Ng, C. K., Piscuoglio, S., Weigelt, B. \u0026amp; Reis-Filho, J. S. 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(2023).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"oncogene","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"onc","sideBox":"Learn more about [Oncogene](http://www.nature.com/onc/)","snPcode":"41388","submissionUrl":"https://mts-onc.nature.com/cgi-bin/main.plex","title":"Oncogene","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-7722676/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7722676/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePKM serves as a rate-limiting enzyme in glycolysis, which produces two isoforms depending on the inclusion of either exon 9 (PKM1) or exon 10 (PKM2). The M2 pyruvate kinase (PKM2) isoform is commonly upregulated in various cancers, where it plays a pivotal role in regulating Warburg effect. Breast cancer stem cells (BCSCs) exhibit enhanced glycolysis, which is crucial for their self-renewal. However, the specific role of PKM2 in BCSCs remains largely unexplored. Here, we report that PKM2 expression is upregulated in BCSCs. Meanwhile, we identify that LINC00887 is significantly upregulated in BRCA through a genome-wide LNCRNA microarray. Moreover, we recognize that hnRNPA1 interacts with PKM pre-mRNA and regulates its mutually exclusive splicing. Furthermore, we demonstrate that LINC00887 maintains the self-renewal of BCSCs by promoting PKM2 splicing and reprogramming glucose metabolism. Mechanistically, LINC00887 upregulates PKM2 expression by binding hnRNPA1, thereby concealing its ubiquitination site, which blocks its ubiquitination and maintains its stability. Consistently, overexpression of hnRNPA1 almost completely rescues/reverses the inhibitory effects of LINC00887 KD in BRCA.Collectively, our study characterizes the LINC00887/hnRNPA1/PKM1/2 axis in BRCA and reveals the essential role of LINC00887 in BCSCs self-renewal/maintenance through promoting hnRNPA1-mediated PKM2 splicing, highlighting the therapeutic potential of targeting cancer metabolism.\u003c/p\u003e","manuscriptTitle":"Blocking ubiquitination of hnRNPA1 maintains the self-renewal of breast cancer stem cells via mutually exclusive splicing of PKM pre-mRNA","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-15 17:06:03","doi":"10.21203/rs.3.rs-7722676/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"revise","date":"2025-10-29T10:41:43+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"This content is not available.","date":"2025-10-26T12:58:54+00:00","index":1,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2025-10-22T12:23:06+00:00","index":2,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2025-10-17T07:34:23+00:00","index":3,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2025-10-13T06:54:35+00:00","index":3,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2025-10-09T07:20:10+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2025-10-05T16:26:13+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewersInvited","content":"","date":"2025-10-02T20:18:05+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-09-29T11:07:30+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-09-26T14:19:59+00:00","index":"","fulltext":""},{"type":"submitted","content":"Oncogene","date":"2025-09-26T14:19:57+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"oncogene","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"onc","sideBox":"Learn more about [Oncogene](http://www.nature.com/onc/)","snPcode":"41388","submissionUrl":"https://mts-onc.nature.com/cgi-bin/main.plex","title":"Oncogene","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"849f9e5f-f59e-471d-aac8-4fde4ceb8297","owner":[],"postedDate":"October 15th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":55688365,"name":"Biological sciences/Stem cells/Cancer stem cells"},{"id":55688366,"name":"Biological sciences/Cancer/Breast cancer"}],"tags":[],"updatedAt":"2026-04-15T07:12:48+00:00","versionOfRecord":{"articleIdentity":"rs-7722676","link":"https://doi.org/10.1038/s41388-026-03776-y","journal":{"identity":"oncogene","isVorOnly":false,"title":"Oncogene"},"publishedOn":"2026-04-14 04:00:00","publishedOnDateReadable":"April 14th, 2026"},"versionCreatedAt":"2025-10-15 17:06:03","video":"","vorDoi":"10.1038/s41388-026-03776-y","vorDoiUrl":"https://doi.org/10.1038/s41388-026-03776-y","workflowStages":[]},"version":"v1","identity":"rs-7722676","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7722676","identity":"rs-7722676","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}