Hypoxia promotes histone H3K9 lactylation to enhance LAMC2 transcription in esophageal squamous cell carcinoma

Summary Hypoxia promotes tumorigenesis and lactate accumulation in esophageal squamous cell carcinoma (ESCC). Lactate can induce histone lysine lactylation (Kla, a recently identified histone marks) to regulate transcription. However, the functional consequence of histone Kla under hypoxia in ESCC remains to be explored. Here, we reveal that hypoxia facilitates histone H3K9la to enhance LAMC2 transcription for proliferation of ESCC. We found that global level of Kla was elevated under hypoxia, and thus identified the landscape of histone Kla in ESCC by quantitative proteomics. Furthermore, we show a significant increase of H3K9la level induced by hypoxia. Next, MNase ChIP-seq and RNA-seq analysis suggest that H3K9la is enriched at the promoter of cell junction genes. Finally, we demonstrate that the histone H3K9la facilitates the expression of LAMC2 for ESCC invasion by in vivo and in vitro experiments. Briefly, our study reveals a vital role of histone Kla triggered by hypoxia in cancer.


Figure
Figure S1.Hypoxia promoted ESCC cells progression and influenced metabolic reprogramming, related to Figure 1.(A) The lactylation of normal esophageal epithelium and ESCC cell lines.(B) The secreted lactic acid levels were increased when KYSE30 cells cultured in hypoxia environment.Bar = 100 μm.(C) Hypoxia significantly increased glucose uptake in KYSE30 cells (n = 3 per group).
Figure S1.Hypoxia promoted ESCC cells progression and influenced metabolic reprogramming, related to Figure 1.(A) The lactylation of normal esophageal epithelium and ESCC cell lines.(B) The secreted lactic acid levels were increased when KYSE30 cells cultured in hypoxia environment.Bar = 100 μm.(C) Hypoxia significantly increased glucose uptake in KYSE30 cells (n = 3 per group).Statistical significance was analyzed by Student's t-test.Data are mean ± SEM. **P < 0.01.

Figure S2 .
Figure S2.The landscape of histone hypoxia lactylation triggered by hypoxia in ESCC cells, related to Figure 2. (A) Histogram showing peptides in KYSE30 cells cultured in hypoxia and normoxia.(B) Sequence motif logo shows a representative sequence for Kla sites.(C) The MS/MS spectra of H3K9la peptides which were marked by 13 C6-lys and 12 C6-lys in hypoxia or normoxia.

Figure S3 .
Figure S3.Genome-wide analysis of the transcriptional consequences of H4K9la in KYSE30 cells cultured in normoxia and hypoxia, related to Figure 3. (A) KEGG enrichment analysis of differentially expressed genes in KYSE30 cells cultured in normoxia and hypoxia.(B-E)GPRC5A, LOXL2, EFEMP2 and ITGA5 promoter in the genomic position was identified to enrich in H3K9la peaks when KYSE30 cells cultured in hypoxia.

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Figure S4.RNA-seq combined with H3K9la MNase ChIP-seq and proteomics analysis in KYSE30 cells cultured in hypoxia, related to Figure 4. (A) Heatmap showed Pearson correlation between hypoxia (H) group RNA-seq data and normoxia (O) group RNA-seq data.(B) KEGG enrichment analysis of differentially expressed genes in KYSE30 cells cultured in normoxia and hypoxia.(C) Reactome enrichment analysis of the overlapped genes.(D)Heatmap showed the collagen-containing extracellular matrix related genes in RNA-seq which promoters in the genomic position was identified to enrich in H3K9la peaks.

Figure S5 .
Figure S5.LAMC2 plays an oncogenic role in ESCC.(A, B) GSEA data showed that LAMC2 mRNA levels was highly expressed in ESCC tumors compared to the normal controls, related to Figure 6.(C) LAMC2 was highly expressed in these ESCC cell lines compared with NE3 cell line.(D) Colony formation assay of KYSE30 cells with overexpression of LAMC2 (n = 3 per group).(E) Transwell assays were used to examine the migratory and invasive abilities of KYSE30 cells with knockdown of LAMC2 (n = 3 per group).(F,G) Wound healing assay show the motility of KYSE30 cells with overexpression of LAMC2.Bar = 100 μm (n = 5 per group).(H) The weight of the tumors was counted (PLVX group = 6, PLVX-3×Flag group = 7).(I) The tumor sizes were continuously recorded to draw tumor growth curves (PLVX group = 6, PLVX-3×Flag group = 7).(J) Metastatic tumor foci in lung were observed (n = 4 per group).(K)Hematoxylin-eosin staining of metastatic tumor foci in lung.The scale bar shows 2 mm.(L) The LAMC2 increased the level of VEGFA and p-AKT in xenograft tumor tissues.(M) Heatmap showed Pearson correlation between LAMC2 overexpressed group RNA-seq data and control group RNA-seq data.(N)Heatmap showed the PI3K-Akt signaling pathway related genes in RNA-seq.