Lnc408 is highly expressed in BCSCs

Our previous study revealed that epithelial–mesenchymal transition (EMT) leads to enhanced stemness characteristic of breast cancer cells21. Using lncRNA array, we identified a series of dysregulated lncRNAs in BCSCs, 21 of which were stemness-associated lncRNAs (Fig. 1A), and some of these lncRNAs were further validated by qRT-PCR (Fig. S1A). Among these lncRNAs, ENST00000422408, termed lnc408 here after, was one of the most highly expressed lncRNAs in BCSCs. To confirm the relationship between lnc408 expression and BCSCs, lnc408 levels were evaluated in CSCs derived from various breast cancer cells, and we found that higher level of lnc408 was in EMT breast cancer cell lines (MDA-MB-468, Hs578T, MDA-MB-231, and BT549) than that in non-EMT cell lines (SKBr3, MDA-MB-453, MCF-7, and T47D). In addition, lnc408 expression was positively correlated with the protein level of stemness marker (e.g., CD44 and SOX2) in both breast cancer cells and clinical samples (Fig. S1B and Fig. 1C).

Fig. 1: Lnc408 is upregulated in BCSCs.
A novel Lnc408 maintains breast cancer stem cell stemness by
recruiting SP3 to suppress CBY1 transcription and increasing
nuclear β-catenin levels

A The heat map showed the major dysregulated stemness-associated lncRNAs in BCSCs derived from epithelial or mesenchymal MCF-7 cells. The red or green dots indicated upregulated or downregulated lncRNAs, respectively. Data are shown as relative fold changes (>2.0) of MCF-7/Twist vs MCF-7/Vector. B Lnc408 expressions were proved by qRT-PCR analysis in non-EMT and EMT BC cells (SKBr3 as a control group, *P < 0.05). C qRT-PCR was used to assess the expression levels of lnc408, CD44, and SOX2 in BC tissues (n = 50). D, E qRT-PCR was conducted to determine the abundances of lnc408 in BCSC derived from BC cell lines and clinical BC samples (**P < 0.01). F Lnc408 expression was determined by qRT-PCR in different passage of BCSCs (P1 to P3; **P < 0.01, ***P < 0.001). For qRT-PCR assays, all data are shown as means ± SD.

CD44+/CD24 has been widely used as BCSC surface markers, thus we sorted CD44+/CD24 subpopulation (termed as BCSCs) from various breast cancer cells and clinical tumor samples, and other remaining subpopulations (CD44+/CD24+, CD44/CD24+, CD44/CD24) were termed as non-BCSCs. The higher level of lnc408 was further confirmed in these CD44+/CD24 subpopulation compared with their non-BCSCs (Figs. 1D, E). Moreover, the gradually elevated lnc408 was observed in the BCSC spheres from first to third generation (Fig. 1F). Kaplan–Meier survival curves showed that patients with high expression of lnc408 had a shorter overall survival than those with low expression of lnc408 (Fig. S1C). These data demonstrate that lnc408 is highly expressed in BCSCs.

Lnc408 is required for self-renewal maintenance of BCSCs

To determine the role of lnc408 in BCSC self-renewal, we silenced lnc408 by separately transfecting two lentivirus-mediated shRNAs into breast cancer cells and primary breast cancer cells derived from clinical samples (Fig. 2A and Fig. S2A). Lnc408 knockdown dramatically reduced sphere formation of breast cancer cells and primary breast cancer cells (Fig. 2B and Fig. S2B). Moreover, the mRNA (Fig. 2C and Fig. S2C) and protein (Fig. 2D and Fig. S2D) levels of representative breast cancer stemness markers, including SOX2, Nanog, and CD44 were notably decreased in the lnc408-knocked down cells compared to their scrambled control cells (sh Ctrl). In line with this, lnc408 knockdown attenuated CD44 and c-Myc expressions in the BCSC spheres checked by IF staining (Fig. S2E). Subsequently, we explored the effects of lnc408 on tumor-initiating capacity using lnc408 wild-type and knocked down primary breast cancer cells isolated from breast cancer tumors, which were subcutaneously injected into mammary fat pad of nude mice. Lnc408 knockdown led to the significant reduce of tumor burden compared with the control primary cancer cells, as detected by a limiting dilution xenograft analysis (Fig. 2E). Furthermore, lnc408 knockdown apparently decreased tumorigenic cell frequency (Fig. 2F). Taken together, these data indicate that lnc408 is required for the maintenance of self-renewal capability of BCSCs.

Fig. 2: Lnc408 is required for the self-renewal maintenance of BCSCs.
A novel Lnc408 maintains breast cancer stem cell stemness by
recruiting SP3 to suppress CBY1 transcription and increasing
nuclear β-catenin levels

A Lnc408 was efficiently silenced by two-independent shRNAs (#2 and #3) in primary BC cells (**P < 0.01). B Mammosphere-forming capacities in primary BC cells with silenced lnc408 were evaluated by suspended culture, and representative images were shown. The right panel represents the statistical results of mammosphere numbers as means ± SD (*P < 0.05; scale bar, 100 μm). C, D BCSC stemness markers (SOX2, Nanog, and CD44) were assessed in lnc408-depleted cells by qRT-PCR (C) and western blotting (D) (*P < 0.05). E Lnc408-depleted or control primary BC cells were transplanted into mammary fat pads of nude mice in tenfold serial dilution manner. Tumor initiation and tumor sizes were monitored over 2 months (N = 6 for each group). F Frequency of tumorigenic cells in lnc408-depleted and scramble primary B cells was analyzed by extreme limiting dilution analysis (#2 clinical sample#2, #5 clinical sample#5, CI confidence interval).

Lnc408 endows non-BCSCs to acquire stemness property

To further clarify the relationship between lnc408 and BCSCs stemness, ectopic lnc408 was stably transfected into non-CSC subpopulation derived from breast cancer cell lines and primary cancer cells (Fig. 3A and Fig. S3A). Compared with non-BCSCs, lnc408 overexpression can restore the non-BCSCs with the sphere formation abilities (Fig. 3B and Fig. S3B). Correspondingly, ectopic lnc408 obviously augmented both mRNA (Fig. 3C and Fig. S3C) and protein (Fig. 3D and Fig. S3D) expressions of breast cancer stemness markers (e.g., SOX2, Nanog, and CD44). Moreover, the effect of ectopic lnc408 on tumorigenesis of non-BCSC population was assessed using limiting dilution experiments in vivo. As few as 1 × 103 of lnc408-overexpressing BT549 cells was sufficient for tumor initiation (2/10 vs 0/10), 1 × 104 of lnc408-overexpressing BT549 cells led to more tumor initiation (4/10 vs 1/10), and injection of 1 × 105 of lnc408-overexpressing BT549 cells resulted in tumorigenesis in most of mice in comparison of non-BCSC cells (8/10 vs 2/10; Fig. 3E), suggesting that lnc408 can enable non-CSCs to acquire stemness characteristic in breast cancer.

Fig. 3: Lnc408 endow non-CSCs to acquire stemness characteristics in breast cancer.
A novel Lnc408 maintains breast cancer stem cell stemness by
recruiting SP3 to suppress CBY1 transcription and increasing
nuclear β-catenin levels

A The expression of ectopic lnc408 was confirmed in non-stemness characteristic breast cancer cells (BT549 and Hs578T; ***P < 0.001; Vec control vector, oeLnc408 overexpress of ectopic lnc408). B Lnc408 overexpression endowed non-CSC breast cancer cells with CSC characteristics, checked by mammosphere formation capacity. The right panel represents the statistical results of mammosphere numbers as means ± SD (*P < 0.05; ND none detected; scale bar, 100 μm). C, D Breast cancer stemness markers (SOX2, Nanog, and CD44) were detected in lnc408-overexpressed cells by qRT-PCR (C) and western blotting (D) (*P < 0.05, **P < 0.01). E Non-CSCs transfected with ectopic Lnc408 or control vector were transplanted into mammary fat pads of nude mice in different cell dose (1 × 103–1 × 105 cells/each mouse), tumorigenicity of nude mice was measured during the experimental period (over 3 months; N = 10 for each group).

Lnc408 suppresses the transcription of CBY1 to maintain BCSCs self-renewal

Next, we asked how lnc408 involves in stemness maintenance of BCSC. It has been reported that lncRNAs can act in cis-regulatory effects to regulate neighboring gene expression25. We found that lnc408 was an antisense lncRNA composed of two exons and spanning 1.08 kilobases (kb), and locating on chromosome 22 near to chibby 1 (CBY1) gene (Fig. 4A). Furthermore, lnc408 was confirmed to be a lncRNA with no coding potentiality (Fig. S4A and Fig. S4B), and mainly distributed in nuclei of breast cancer cells checked by RNA-FISH assay (Fig. 4B).

Fig. 4: Lnc408 regulates the expression of target gene CBY1.
A novel Lnc408 maintains breast cancer stem cell stemness by
recruiting SP3 to suppress CBY1 transcription and increasing
nuclear β-catenin levels

A Schematic annotation of lnc408 genomic locus on chromosome 22. Black rectangles represent exons. B Intracellular localization of lnc408 was visualized in BC cells by RNA-FISH assays. Representative images of lnc408 in BT549 cells are shown. U6 served as a nucleus control and 18S as a cytoplasm control; cell nuclei were counterstained with DAPI (scale bar, 10 μm). C Full length of lnc408 transcripts (sense), antisense transcripts and control probe were labeled with biotin, then RNA-pull down was performed using nuclear extracts of mammospheres derived from BT549 cells. The interesting bands were followed by mass spectrometry. The arrow indicates the target of SP3. D The lnc408-binding protein SP3 in RNA-pull down precipitates (as carried out as in C, GAPDH as a negative control) was further confirmed by western blotting. E The interaction between lnc408 and SP3 was verified by an RNA immunoprecipitation (RIP) assay (**P < 0.01). Data are shown as means ± SD. F, G The mRNA or protein expression of CBY1 was determined by qRT-PCR (F) and western blotting (G) in lnc408-silenced BCSCs, respectively (*P < 0.05, **P < 0.01). H, I The mRNA and protein expressions of CBY1 were determined by qRT-PCR (H) and western blotting (I) in SP3-knocked down BCSCs, respectively (**P < 0.01).

Since lncRNAs could exert their functions through interacting with proteins to regulate gene expression in the nuclei26, an RNA pull-down assay with biotin-labeled lnc408 was performed to seek the potential lnc408-associated proteins. SP3 was identified to bind with lnc408 in BCSCs (Fig. 4C, D), and the interaction of lnc408 with SP3 was further validated by immunoprecipitation (RIP; Fig. 4E). In addition, we confirmed that lnc408 knockdown promoted CBY1 mRNA (Fig. 4F) and protein (Fig. 4G) expression in BCSCs. Similarly, efficient silence of SP3 (Fig. S4C, D) led to the enhanced mRNA (Fig. 4H) and protein (Fig. 4I) levels of CBY1, which is consistent with the finding that SP3 could serve inhibitory transcription function27,28. These data suggest that lnc408 recruits SP3 to suppress the transcription of CBY1.

To investigate the role of CBY1 in the self-renewal of BCSC, ectopic CBY1 was efficiently transfected into BCSCs (Fig. S5A, B). As shown in Fig. 5A, CBY1 overexpression markedly reduced sphere number and their average diameter. Correspondingly, ectopic CBY1 significantly decreased SOX2, Nanog, and CD44, the representative breast cancer stemness marker expressions both in mRNA (Fig. S5C) and protein (Fig. 5B) levels, implying that CBY1 has the ability to inhibit the stemness of BCSCs. To further verify the function of CBY1 in stemness maintenance of BCSCs, CBY1 was transfected into non-BCSC with ectopic lnc408, and mammosphere formation and CSC-associated markers were assessed. As shown, the ectopic lnc408-induced mammosphere formation potentials (Fig. 5C) and stemness-related protein expressions (Fig. 5D, E) were notably decreased by CBY1. Besides, the expression of CBY1 was negatively related with the levels of CD44 and SOX2, two of known stemness biomarkers, in our cohort of clinical samples (Fig. S5D), these findings were further confirmed in large samples downloaded from Metabric database (Fig. 5F). Indeed, the breast cancer patients with high level of CBY1 had a higher overall survival in comparison with these with low level of CBY1 (Fig. 5G). Overall, lnc408 may involve in maintenance of BCSCs by inhibiting CBY1.

Fig. 5: Lnc408 suppresses the expression of CBY1 to regulate BCSC self-renewal and stemness maintenance.
A novel Lnc408 maintains breast cancer stem cell stemness by
recruiting SP3 to suppress CBY1 transcription and increasing
nuclear β-catenin levels

A Ectopic CBY1 was transfected into BC cells to check the mammosphere-forming capacity of CSCs, and representative pictures of mammospheres are shown. The right panel represents the statistical results of mammosphere numbers as means ± SD (*P < 0.05; scale bar, 100 μm). B Breast cancer stemness markers (SOX2, Nanog, and CD44 were assessed in CBY1-overexpressed BCSCs by western blotting. C The non-CSC-like BC cells were transfected with control vector, lnc408 or lnc408 combined with CBY1, the mammosphere formation capacities were assessed in serum-free suspended culture. The right panel represents the statistical results of mammosphere numbers as means ± SD (**P < 0.01; ND none detected; scale bar, 100 μm). D, E The mRNA and protein levels of CSC markers (SOX2, Nanog, and CD44) were determined by qRT-PCR (D) and western blotting (E), respectively (*P < 0.05, **P < 0.01). F Pearson correlation analysis of CBY1 and CD44, CBY1 and SOX2 in 474 breast cancer tissues based on Metabric database. G Kaplan–Meier survival analysis to show the BC patients with low CBY1 expression (N = 3851) had poor prognosis, using TCGA database.

Lnc408-mediated decrease of CBY1 activates Wnt/β-catenin signaling

Subsequently, we wondered how lnc408-mediated CBY1 involves in regulating the stemness of BCSCs. By using bioinformatics analysis, we found CBY1 could interact with 14-3-3, AKT, β-catenin, and other proteins (Fig. 6A). It has been reported that the phosphorylation of CBY1 by ATK lead to an activated CBY1, which binds with 14-3-3 and β-catenin to form a ternary complex, and causing β-catenin to be pumped out of nuclei29. Agreeing with the previous findings, we confirmed that CBY1 could interact with 14-3-3 and β-catenin to form a ternary complex, mutation of CBY1 at 20 sites from serine into alanine (mutant CBY1 S20A) caused the disintegration of the complex (Fig. 6B) in 293T cells. Indeed, the trimer of CBY1/14-3-3/β-catenin was mainly existed in non-BCSCs rather than in BCSCs, which could be abrogated when either ectopic lnc408 was transfected into non-BCSC or CBY1 was silenced in non-BCSC (Fig. 6C, left panel). However, restoration of CBY1 expression or knockdown of lnc408 in BCSC facilitated the ternary complex formation (Fig. 6C, right panel). Consistently with the ternary complex formation in non-BCSCs or disintegration in BCSCs, higher level of cytoplasmic β-catenin than nuclear β-catenin was detected in non-BCSCs, the distribution of cytoplasmic or nuclear β-catenin in non-BCSC could be reversed by overexpression of lnc408 or knockdown of CBY1 (Fig. 6D, left panel); however, the lower level of cytoplasmic β-catenin and higher level of nuclear β-catenin in BCSCs could also be reversed by ectopic CBY1 or silence of lnc408 (Fig. 6D, right panel). Correspondingly, CBY1-mediated the ternary complex formation and nuclear β-catenin accumulation were closed with the change of mammosphere formation capability in BCSC derived from BT549 cells (Fig. 6E), in which ectopic CBY1 induced decrease of nuclear β-catenin blunted mammosphere formation, SKL2001 (an agonist of Wnt signaling) treatment rescued the spheres formation ability (Fig. 6E). In addition, previous study has found that stemness-related gene c-Myc and Klf4 were at downstream of Wnt/β-catenin signaling in bovine embryonic stem cells (ESCs)30. Similarly, the same trend of stemess-related gene expression, including c-Myc and Klf4 was observed in these BCSCs (Fig. 6F, G). These data demonstrate that lnc408-mediated decrease of CBY1 stimulates Wnt/β-catenin signaling and acts a pivotal role in stemness maintenance of BCSCs.

Fig. 6: Lnc408-mediated CBY1 decrease triggers WNT signaling and promotes self-renewal and stemness maintenance of BCSCs.
A novel Lnc408 maintains breast cancer stem cell stemness by
recruiting SP3 to suppress CBY1 transcription and increasing
nuclear β-catenin levels

A A protein networks showing the potential interaction among 14-3-3 (YWHAZ), β-catenin (CTNNB1), and CBY1. B HEK293T cells were transfected with an expression vector encoding wild-type CBY1 (WT) or mutant CBY1 (S20A). Immunoprecipitation and western blotting were conducted with antibodies against 14-3-3, β-catenin, and CBY1 to detect the interaction of CBY1, 14-3-3, and β-catenin. C IP-WB was used to detect the ternary complex of CBY1, 14-3-3, and β-catenin in non-CSC derived from of BT549, non-CSC transfected with ectopic lnc408 (oelnc408), and non-CSC with silenced CBY1 (sh-CBY1); or in CSC derived from BT549, CSC transfected with ectopic CBY1 (oeCBY1), and CSC with silenced lnc408 (sh-lnc408). D Western blotting to check the cellular distribution of β-catenin as in C (C cytoplasm, N nucleus). GAPDH served as a cytoplasm control, and PCNA as a nucleus control. E The mammosphere-forming abilities of BT549, BT549 transfected with CBY1 (oeCBY1), or BT549/ocCBY1 cells treated with or without SLK2001 (WNT/β-catenin agonist, 10 μM) were tested under suspended culture. The down panel shows the statistical results of mammosphere numbers as means ± SD (**P < 0.01; NS none significance; scale bar, 100 μm). F, G The mRNA and protein expressions of breast cancer pluripotent factors (KLF4, c-Myc) were determined by qRT-PCR (F) and western blotting (G), respectively (*P < 0.05; **P < 0.01; NS none significance).

Lnc408/CBY1 axis facilitates BCSC tumorigenesis in vivo

Lastly, the effect of lnc408/CBY1 axis on tumorigenesis and tumor growth was assessed in vivo by injection of lnc408-knocked down BCSCs and lnc408-overexpressing non-BCSCs into female nude mice. As expectedly, in comparison with BCSC with high level of endogenous lnc408, knockdown of lnc408 notably decreased tumorigenesis and tumor growth; while ectopic lnc408 in the non-BCSCs significantly promoted tumorigenesis and increased tumor growth, and the lnc408-droved tumor initiation was severely blunted by reexpression of CBY1 in non-BCSC (Fig. 7A). The changes of c-MYC protein, another known stemness biomarker, were detected in the corresponding tumor tissues (Fig. 7B). Meanwhile, nuclear β-catenin protein, the downstream target of lnc408/CBY1 axis, was significantly increased in the tumor derived from injected BCSC (BCSC/shCtrl) compared to the tumor from injected non-BCSCs (non-BCSC/Vec), and it was reduced in tumor derived from lnc408-silenced BCSCs (Fig. 7C). The mice injected with lnc408-overexpressing non-BCSC had high rate of tumor initiation (Fig. 7A), which was compatible with the increased expression of nuclear β-catenin in the tumor (Fig. 7C); the mice injected with non-BCSC transfected with both ectopic lnc408 and CBY1 (non-BCSC/lnc408/CBY1) just had few tumor initiation (Fig. 7A), due to lowest nuclear β-catenin, which accompanied with high level of phosphorylated cytoplasm β-catenin, in the tumor (Fig. 7C), suggesting an essential role of lnc408/CBY1 axis in promoting tumor initiation of BCSCs via regulation of β-catenin signaling. To expand this finding, we assessed the lnc408 expression and the corresponding nuclear β-catenin and c-Myc protein levels in clinical tumor tissues. As shown in Fig. 7D, the enhanced nuclear β-catenin (n-β-catenin) and c-MYC (the representative stemness-related marker protein) accompanied with decreased phosphorylated cytoplasm β-catenin were detected in the randomly selected lnc408 high expressed tumors in comparison with the lnc408 low expressed tumors (Fig. 7D). In conclusion, in non-BCSCs, CBY1 forms ternary complex with 14-3-3 and β-catenin to result in the complex being bumped out of nucleus and the degradation of β-catenin; in BCSCs, lnc408 recruits SP3 to cause the transcription depression of CBY1, which prevents the formation of CBY1/14-3-3/β-catenin complex, and β-catenin is accumulated in the nucleus to promote the self-renewal and stemness of BCSCs (Fig. 7E).

Fig. 7: Lnc408/CBY1 axis promotes tumorigenesis of BCSCs in vivo.
A novel Lnc408 maintains breast cancer stem cell stemness by
recruiting SP3 to suppress CBY1 transcription and increasing
nuclear β-catenin levels

A The tumor initiation and tumor sizes of each group (*P < 0.05, **P < 0.01). B Representative pictures of IHC staining of c-Myc proteins (upper panel) and c-Myc mRNA levels (down panel) in each group (*P < 0.05; scale bar, 50 μm). C Protein levels of nuclear, phosphorylated, and total β-catenin in tumors were determined by western blotting. D Clinical samples were divided into lnc408 high and low expression groups according to lnc408 expression levels, and subjected to western blotting to assess nuclear β-catenin, total β-catenin, and c-Myc expressions. E A schematic model to illustrate lnc408 molecular functions in BCSCs. Lnc408 recruits SP3 to impair CBY1 transcription, the reduced CBY1 lose or significantly incapacitated its ability to form complex with 14-3-3 and β-catenin in CSCs, which notably mitigate the transportation of β-catenin into cytoplasm, to lead an accumulation of nucleus β-catenin, thus triggers the WNT/β-catenin signaling to maintain BCSCs stemness.

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