Exosomal lncRNA Mir100hg from lung cancer stem cells activates H3K14 lactylation to enhance metastatic activity in non-stem lung cancer cells

Abstract

The mean survival of metastatic lung adenocarcinoma is less than 1 year, highlighting the urgent need to understand the mechanisms underlying its high mortality rate. The role of Extracellular vesicles (EVs) in facilitating the interactions between cancer cells and the metastatic microenvironment has garnered increasing attention. Previous studies on the role of EVs in metastasis have been primarily focused on cancer cell-derived EVs in modulating the functions of stromal cells. However, whether cancer stem cells (CSCs) can alter the metastatic properties of non-CSC cells, and whether EV crosstalk can mediate such interaction, have not been demonstrated prior to this report. In the present study, we integrated multi-omics sequencing and public database analysis with experimental validation to demonstrate, for the first time, the exosomal Mir100hg, derived from CSCs, could enhance the metastatic potential of non-CSCs both in vitro and in vivo. Mechanistically, HNRNPF and HNRNPA2B1 directly binds to Mir100hg, facilitating its trafficking via exosomes to non-CSCs. In non-CSCs, Mir100hg upregulates ALDOA expression, subsequently leading to elevated lactate production. Consequently, the increased lactate levels enhance H3K14 lactylation by 2.5-fold and promote the transcription of 169 metastasis-related genes. This cascade of events ultimately results in enhanced ALDOA-driven glycolysis and histone lactylation-mediated metastatic potential of non-CSC lung cancer cells. We have delineated a complex regulatory network utilized by CSCs to transfer their high metastatic activity to non-CSCs through exosomal Mir100hg, providing new mechanistic insights into the communication between these two heterogeneous tumor cell populations. These mechanistic insights provide novel therapeutic targets for metastatic lung cancer, including HNRNPF/HNRNPA2B1-mediated Mir100hg trafficking and the histone lactylation pathway, advancing our understanding of CSC-mediated metastasis while suggesting promising strategies for clinical intervention.

Keywords: ALDOA; Exosomal lncRNA; H3K14; Histone lactylation; Mir100hg.

Fig. 1

HNRNPF interacts with Mir100hg and promotes its localization within the cytoplasm. A Schematic diagram of exosome-based animal experiments; B Fluorescence imaging of mouse lung tumor (green) and hematoxylin and eosin (HE) stained lung section; C Nucleoplasmic RNA isolation from LLC and LLC-SD cells; D Fluorescence in situ hybridization (FISH) of Mir100hg in LLC and LLC-SD cells; E RNA pulldown assay for Mir100hg; F Venn diagram of RNA pulldown results; G Table of information related to HNRNP family protein profiles in RNA-pulldown results; H Nuclear and cytoplasmic RNA isolation from LLC-SD after knocking down HNRNP family proteins; I FISH analysis of Mir100hg; J RNA immunoprecipitation (RIP) for Mir100hg and HNRNPF; K RNA pulldown for Mir100hg and HNRNPF; L Fluorescence co-localization of Mir100hg and HNRNPF. M Schematic representation of direct binding of Mir100hg to HNRNPF for nucleoplasmic translocation. (*p < 0.05, **p < 0.01, ***p < 0.001)

Fig. 2

HNRNPA2B1 enhances the metastatic potential of LLC by facilitating the incorporation of Mir100hg into exosomes. A Schematic diagram of exosomal Mir100hg tracing experiment; B Co-localization of Mir100hg and exosomes in LLC cells; C Bar graph showing differential expression of Mir100hg in LLC cells post-exosome incubation; D Mir100hg expression in LLC-SD exosomes following HNRNP family protein knockdown; E RNA immunoprecipitation (RIP) for Mir100hg and HNRNPA2B1; F RNA pulldown for Mir100hg and HNRNPA2B1; G Mir100hg expression in LLC-SD cells following HNRNPA2B1 knockdown; H Mir100hg expression in LLC cells co-cultured with exosomes from HNRNPA2B1-knockdown LLC-SD cells; I, J Transwell for LLC cells post-co-culture with LLC-SD exosomes and the counting chart. (*p < 0.05, **p < 0.01, ***p < 0.001)

Fig. 3

Mir100hg promotes the metastasis of lung cancer cells in vitro and in vivo by up-regulating ALDOA expression. A Protein profile of ALDOA in RNA-PULLDOWN; B ALDOA expression differences in TCGA-LUAD database; C Line graph of the effect of ALDOA on overall survival in TCGA-LUAD database; D Analysis of ALDOA protein levels in Mir100hg knockdown (sh-Mir100hg) and overexpression cells (OE-Mir100hg); E, F Transwell of the migration and invasion ability of LLC cells and the counting chart; G Lung tumors in various mouse groups (green); H Hematoxylin and eosin (HE) stained lung sections (arrows indicate tumor areas); I Schematic diagram of ALDOA involvement in glycolysis; J Transwell migration and invasion assays post-addition of GADP in ALDOA knockdown cells and the counting chart. (*p < 0.05, **p < 0.01, ***p < 0.001)

Fig. 4

Mir100hg positively regulates ALDOA mRNA and protein levels through CeRNA and OTUD4-deubiquitination. A RNA immunoprecipitation (RIP) for Mir100hg and ALDOA; B RNA pulldown for Mir100hg and ALDOA; C Analysis of ALDOA protein levels post-MG132 treatment in Mir100hg knockdown and overexpression cells; D Analysis of ALDOA protein levels and relative band intensity quantification at indicated time points post-CHX treatment in Mir100hg knockdown and overexpression cells; E Ubiquitination levels of pulldown ALDOA protein analyzed by Western blot in Mir100hg knockdown and overexpression cells; F Protein spectrum results for OTUD4; G Ubiquitination analysis of ALDOA protein pulldown in OTUD4 knockdown cells by Western blot; H, I Analysis of OTUD4 levels following ALDOA protein pulldown in Mir100hg knockdown cells and Analysis of OTUD4 levels following ALDOA protein pulldown in Mir100hg knockdown cells; J, K RNA immunoprecipitation (RIP) and RNA-PULLDOWN for Mir100hg and OTUD4; L Fluorescence co-localization of Mir100hg with OTUD4 and ALDOA; M Schematic representation of ALDOA ubiquitination inhibition by Mir100hg through OTUD4 (*p < 0.05, **p < 0.01, ***p < 0.001)

Fig. 5

Mir100hg facilitates lung cancer cell metastasis by enhancing lactylation. A Metabolomics heatmap; B Global lactylation and H3K14la levels detected by Western blot (WB); C, D Immunofluorescence detection of global lactylation and H3K14la levels; E Schematic diagram illustrating glycolytic steps affected by various drugs and inhibitors; F Global lactylation and H3K14la levels in OE-Mir100hg cells post-GNE treatment and subsequent NALA exposure; G Transwell of migration and invasion ability of LLC cells after si-Ldha and the counting bar chart. H Transwell migration and invasion assays of LLC cells post-GNE and NALA and the counting bar chart (*p < 0.05, **p < 0.01, ***p < 0.001)

Fig. 6

Multi-omics analysis reveals the involvement of the Mir100hg-H3K14la axis in the transcription of tumor metastasis-related genes. A Bar graph showing the number of H3K14la peaks enriched across four groups; B Heatmap of H3K14la enrichment in TSS regions across four groups; C Enrichment of H3K14la in TSS regions of genes exhibiting varied expression levels in RNA-Seq; D Differential chromatin opening in H3K14la-enriched genes within TSS regions; E Differential heatmap in RNA-Seq of upregulated genes in OE-Mir100hg and corresponding TSS region heatmap in CUT&Tag; F GSEA analysis indicating enrichment of differentially expressed genes in OE-Mir100hg within tumor metastasis-related gene set; G Pie chart of the percentage of H3K14la-enriched genes among upregulated genes in OE-Mir100hg group; H KEGG analysis of pathways associated with H3K14LA-enriched upregulated genes; I–L Display of genes rescued post-addition of GNE in OE-Mir100hg group; M Ptgs2 peaks identified in RNA-Seq and CUT&Tag; N Heatmap of TSS region enrichment for tumor metastasis-related genes in CUT&Tag and log fold change (logFC) heatmap in RNA-Seq (*p < 0.05, **p < 0.01, ***p < 0.001)

Fig. 7

The Mir100hg-H3K14 lactylation axis enhances the expression of genes associated with tumor metastasis. A Flow chart for gene screening process; B Boxplot of LMAN1, OSTC, P4HA1, TEX30, and TMEM65 gene expression differences in TCGA-LUAD; C Lman1/Ostc/P4ha1/Tex30/Tmem65 in CUT&Tag peak figure display, mRNA expression changes and survival analysis; D Transwell for cell migration and invasion post-knockdown of LMAN1, OSTC, P4HA1, TEX30, and TMEM65 and the counting bar chart (*p < 0.05, **p < 0.01, ***p < 0.001)