Dissection of FOXO1-Induced LYPLAL1-DT Impeding Triple-Negative Breast Cancer Progression via Mediating hnRNPK/β-Catenin Complex

Abstract

Triple-negative breast cancer (TNBC) is considered as the most hazardous subtype of breast cancer owing to its accelerated progression, enormous metastatic potential, and refractoriness to standard treatments. Long noncoding RNAs (lncRNAs) are extremely intricate in tumorigenesis and cancerous metastasis. Nonetheless, their roles in the initiation and augmentation of TNBC remain elusive. Here, in silico analysis and validation experiments were utilized to analyze the expression pattern of clinically effective lncRNAs in TNBC, among which a protective lncRNA LYPLAL1-DT was essentially curbed in TNBC samples and indicated a favorable prognosis. Gain- and loss-of-function assays elucidated that LYPLAL1-DT considerably attenuated the proliferative and metastatic properties along with epithelial-mesenchymal transition of TNBC cells. Moreover, forkhead box O1 (FOXO1) was validated to modulate the transcription of LYPLAL1-DT. Mechanistically, LYPLAL1-DT impinged on the malignancy of TNBC mainly by restraining the aberrant reactivation of the Wnt/β-catenin signaling pathway, explicitly destabilizing and diminishing β-catenin protein by interacting with heterogeneous nuclear ribonucleoprotein K (hnRNPK) and constricting the formation of the hnRNPK/β-catenin complex. Conclusively, our present research revealed the anti-oncogenic effects of LYPLAL1-DT in TNBC, unraveling the molecular mechanisms of the FOXO1/LYPLAL1-DT/hnRNPK/β-catenin signaling axis, which shed innovative light on the potential curative medicine of TNBC.

Fig. 1.

LYPLAL1-DT expression is curbed in TNBC samples and correlates with a favorable prognosis. (A) Venn diagram depicting the identification of 73 common differentially expressed lncRNAs from GSE115275, GSE119233, and TCGA-TNBC cohorts. (B) Heatmaps showing the expression patterns of 73 candidate lncRNAs in three datasets. (C) Univariate Cox regression analysis based on 73 eligible lncRNAs in TCGA-TNBC dataset. (D) Expression level of LYPLAL1-DT in TNBC and nontumor normal samples of three datasets. (E) Kaplan–Meier curves displaying TNBC patients with high expression of LYPLAL1-DT gain favorable overall survival, progression-free survival, and disease-specific survival. (F) Expression level of LYPLAL1-DT in tumor and nontumor normal samples across various cancer types in the TCGA cohort, including breast cancer (BRCA). (G) Expression level of LYPLAL1-DT in 120 matched breast cancer (BC) and noncancerous normal samples of the TCGA-BRCA cohort. (H) Relative expression level of LYPLAL1-DT in 24 pairs of TNBC and matched nontumor tissues of the SYSUCC cohort using RT-qPCR. (I) Expression level of LYPLAL1-DT in TNBC patients with various T stage, N stage, or AJCC stage from the TCGA-TNBC cohort. (J) Multivariate Cox regression analysis incorporating age, T stage, N stage, AJCC stage, and LYPLAL1-DT. Error bars represent mean ± SD. *P < 0.05, **P < 0.01, ****P < 0.0001.

Fig. 2.

LYPLAL1-DT is transcriptionally modulated by FOXO1.(A) Relative expression level of LYPLAL1-DT in TNBC cell lines, non-TNBC cell lines, and MCF10A. (B) Venn diagram delineating the identification of potential transcription factors (TFs) regulating LYPLAL1-DT. (C) Binding probability between the eligible TFs and the promoter of LYPLAL1-DT calculated in the JASPAR database. (D) Expression level of FOXO1 in nontumor normal samples and TNBC samples in the TCGA-TNBC cohort. (E) Kaplan–Meier curve showing that breast cancer patients with low expression of FOXO1 attain an inferior overall survival. (F) Expression level of FOXO1 protein (top) and FOXO1 RNA (bottom) in TNBC cell lines and MCF10A. (G) Significant positive correlation between LYPLAL1-DT and FOXO1 expression levels in the TCGA-TNBC cohort. (H) Validation of silencing FOXO1 in MDA-MB-231 and BT-549 cells and overexpressing FOXO1 in MDA-MB-157 cells (top), in addition with the corresponding expression alterations of LYPLAL1-DT (bottom). (I) ChIP-seq from the ENCODE database verifying FOXO1 binding peaks on the promoter region of LYPLAL1-DT in HepG2 cells. (J) The binding motif of FOXO1 (left) and the predicted binding site of FOXO1 on the promoter region of LYPLAL1-DT as well as the designed fragments for the further ChIP analysis (right). (K) Validation of FOXO1 antibody utilized in IP assay (top) and RT-qPCR result for ChIP assay using antibodies against FOXO1 and IgG (bottom). (L) Confirmation of RT-qPCR products in ChIP assay using nuclear acid electrophoresis. (M) Promoter dual-luciferase reporter assay conducted by cotransfections of plasmid pEZX-FR01 inserting LYPLAL1-DT promoter and plasmid overexpressing FOXO1 (FOXO1), or siRNA knocking down FOXO1 (siFOXO1), or the corresponding control plasmid (Scramble) or nontarget siRNA (siCtrl), which unraveled that FOXO1 definitely bound with the promoter of LYPLAL1-DT. Error bars represent mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig. 3.

LYPLAL1-DT exerts antitumorigenic and antimetastatic effects to TNBC cells in vitro. (A) RT-qPCR result validating effects of overexpressing LYPLAL1-DT in MDA-MB-231 and BT-549 cells and knocking down LYPLAL1-DT in SUM159PT cells. (B) CCK-8 proliferation assays manifesting that overexpressing LYPLAL1-DT limited the proliferation of TNBC cells, while silencing LYPLAL1-DT promoted the proliferative nature. (C to F) Representative graphs of EdU staining proliferation assays (C), colony formation assays (D), transwell assays (E), and wound-healing assays (F) after performing overexpression or knockdown of LYPLAL1-DT in TNBC cells. (G) Quantitative data of EdU staining proliferation assays (top) and colony formation assays (bottom). (H) Quantitative data of transwell assays evaluating migration (top) or invasion (medium) properties and wound-healing assays (bottom). (I) Markers of epithelial-mesenchymal transition (EMT) were investigated in condition of overexpressing or knocking down LYPLAL1-DT in TNBC cells. Error bars represent mean ± SD. Scale bars in this figure all signify 100 μm. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig. 4.

LYPLAL1-DT obstructs tumor growth and lung metastasis of TNBC cells in vivo. (A to C) Representative images of xenograft tumor morphology (A), quantitative data of tumor growth (B), and tumor weights of xenograft tumors (C) in nude mice (five mice per group) injected subcutaneously into the mammary fat pad using the corresponding stably transfected TNBC cells. (D to I) Representative images of optical luciferase imaging assays in vivo (D and E) and lung metastatic morphology and H&E staining in lung metastatic foci (F and G), as well as quantitative data of optical luciferase imaging assays (H) and metastatic foci in lung tissue (I) in nude mice (five mice per group) injected intravenously via the tail vein using the corresponding stably transfected TNBC cells. Error bars represent mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig. 5.

LYPLAL1-DT inhibits the Wnt/β-catenin pathway by enhancing ubiquitination of β-catenin and thereby destabilizing its protein. (A) Subcellular fractionation assays showing the relative abundance of LYPLAL1-DT in the cytoplasm and nucleus of TNBC cells. GAPDH and U6 were applied as positive controls for the cytoplasmic and nuclear fractions, respectively. (B) RNA-FISH depicting the translocation of LYPLAL1-DT in the cytoplasm and nucleus of TNBC cells (scale bar, 20 μm). 18S and U6 were applied as positive controls for the cytoplasmic and nuclear fractions, respectively. (C and D) Functional enrichment assays using KEGG pathways (C) and GO pathways (D) based on the coding genes highly correlated to LYPLAL1-DT. (E) GSEA analysis illustrating that the expression of LYPLAL1-DT was negatively related to the Wnt signaling pathway but positively associated with ubiquitin-mediated proteolysis. (F) Expression levels of β-catenin and its downstream oncogenic genes were detected in TNBC cells ectopically overexpressing or silencing LYPLAL1-DT. (G) Results of TOP/FOP-flash luciferase assays indicating the transcriptional activity of β-catenin after increasing or decreasing LYPLAL1-DT expression. (H) RT-qPCR results confirming the relative RNA alterations of β-catenin and its downstream oncogenic genes when overexpressing LYPLAL1-DT. (I) Subcellular expression of β-catenin protein in the cytoplasm and nucleus was assessed after up-regulating or down-regulating LYPLAL1-DT in TNBC cells. β-Tubulin and H3 histone were administrated as positive controls for the nuclear and cytoplasmic fractions, respectively. (J) Immunocytochemistry staining showing β-catenin accumulation in the nucleus after overexpressing or knocking down LYPLAL1-DT in TNBC cells (scale bar, 50 μm). (K) CHX-chase assay performed in TNBC cells overexpressing LYPLAL1-DT and meanwhile treated with CHX (50 μg/ml) at 0, 2, 4, and 6 h to observe β-catenin expression. (L) Western blot showing the expression of β-catenin in LYPLAL1-DT-overexpressed or control TNBC cells treated with or without CHX and MG132 (25 μM) at 6 h. (M and N) Ubiquitination of β-catenin was investigated in input samples (left) and IP samples (right). Error bars represent mean ± SD. **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig. 6.

LYPLAL1-DT regulates ubiquitination of β-catenin by interacting with hnRNPK. (A) Result of RNA pull-down analysis was displayed by silver staining (left) and mass spectrometry (right). (B) Western blot for RNA pull-down analysis validating the interplay between LYPLAL1-DT and hnRNPK. (C) Validation of hnRNPK antibody used in IP assay (top) and results of RIP assay (bottom) to reconfirm the direct connection between LYPLAL1-DT and hnRNPK. (D) A schematic diagram of full-length of LYPLAL1-DT and specific exon fragments (top) and Western blot for RNA pull-down analysis using different biotinylated RNA fragments of LYPLAL1-DT (bottom). (E) A schematic picture of full domains and constructed flag-tagged truncations of hnRNPK (top) and Western blotting analysis to evaluate transfections of flag-tagged truncations of hnRNPK (medium), as well as results of RIP assays using anti-flag antibody (bottom). (F and G) Expression level of hnRNPK in whole TNBC cells (F) and in the cytoplasm or nucleus (G) after elevating or diminishing LYPLAL1-DT expression. (H) Western blotting analysis for β-catenin and hnRNPK in the IP samples with antibodies against hnRNPK or IgG in the presence or absence of overexpressed LYPLAL1-DT. (I) Ubiquitination of β-catenin was discovered in input samples (left) and IP samples using antibody against HA (right) after cotransfections under guidance of the designed groups. (J) Western blotting analyses showing that LYPLAL1-DT partly attenuated hnRNPK-induced accumulation of β-catenin in TNBC cells. (K) TOP/FOP-flash luciferase assays indicating that LYPLAL1-DT partially limited hnRNPK-generated transcriptional activity of β-catenin. (L) Representative IF images displaying alterations of colocalizations between β-catenin and hnRNPK in LYPLAL1-DT-overepressed BT-549 cells (top) or MDA-MB-231 cells (medium) and LYPLAL1-DT-silenced SUM159PT cells (bottom) (scale bar, 20 μm). (M and N) Results of CCK-8 proliferation assay (M) and quantitative data of wound-healing assays (N) after conducting cotransfections by hnRNPK plasmid and LYPLAL1-DT plasmid. Data are mean ± SD. *P < 0.05, ****P < 0.0001.

Fig. 7.

LYPLAL1-DT functions as a tumor suppressor partly by restraining aberrant reactivation of β-catenin. (A) Expression levels of β-catenin, markers of EMT, and β-catenin-activated downstream oncogenic genes were detected in stably LYPLAL1-DT-overexpressed or the control BT-549 and MDA-MB-231 cells with transfection with CTNNB1 (encoding β-catenin) plasmid or the control plasmid according to the instructions of the designed groups. (B to F) Result of CCK-8 proliferation assays (B), representative graphs of EdU staining proliferation assays (C), colony formation assays (D), transwell assays (E), and wound-healing assays (F) conducted in the designed groups above were displayed. Scale bars represent 100 μm. (G) Quantitative data of EdU staining proliferation assays, colony formation assays, transwell assays evaluating migration or invasion ability, and wound-healing assays. (H and I) Image of xenograft tumor morphology (H) and quantitative data of tumor weights of xenograft tumors (I) in nude mice (4 mice per group) injected subcutaneously into the mammary fat pad using stably LYPLAL1-DT-overexpressed MDA-MB-231 cells or stably transfected MDA-MB-231 cells simultaneously overexpressing LYPLAL1-DT and β-catenin or the control cells. Data are mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig. 8.

LYPLAL1-DT conserves as a novel target for TNBC treatment. (A) Representative images of H&E staining and IHC analyses using hnRNPK, β-catenin, Ki-67, E-cadherin, N-cadherin, vimentin, and ZEB1 antibodies in tissues of the xenograft tumors developed by stably LYPLAL1-DT-overexpressed BT-549 or MDA-MB-231 cells and the corresponding control cells in nude mice. (B) The expression of β-catenin, Ki-67, E-cadherin, N-cadherin, vimentin, and ZEB1 by IHC H-score in Fig. 8A. (C) Schematic view illustrating the functional role and regulatory mechanisms of LYPLAL1-DT in suppressing TNBC progression. Data are mean ± SD. **P < 0.01, ***P < 0.001, ****P < 0.0001.