A druggable cascade links methionine metabolism to epigenomic reprogramming in squamous cell carcinoma

Abstract

Upper aerodigestive squamous cell carcinoma (UASCC) is a common and aggressive malignancy with few effective therapeutic options. Here, we investigate amino acid metabolism in this cancer, surprisingly noting that UASCC exhibits the highest methionine level across all human cancers, driven by its transporter LAT1. We show that LAT1 is also expressed at the highest level in UASCC, transcriptionally activated by UASCC-specific promoter and enhancers, which are directly coregulated by SCC master regulators TP63/KLF5/SREBF1. Unexpectedly, unbiased bioinformatic screen identifies EZH2 as the most significant target downstream of the LAT1-methionine pathway, directly linking methionine metabolism to epigenomic reprogramming. Importantly, this cascade is indispensable for the survival and proliferation of UASCC patient-derived tumor organoids. In addition, LAT1 expression is closely associated with cellular sensitivity to inhibition of the LAT1-methionine-EZH2 axis. Notably, this unique LAT1-methionine-EZH2 cascade can be targeted effectively by either pharmacological approaches or dietary intervention in vivo. In summary, this work maps a unique mechanistic cross talk between epigenomic reprogramming with methionine metabolism, establishes its biological significance in the biology of UASCC, and identifies a unique tumor-specific vulnerability which can be exploited both pharmacologically and dietarily.

Keywords: LAT1; UASCC; cancer metabolism; epigenomics; methionine.

Fig. 1.

UASCC displays uniquely high LAT1/methionine levels across human cancers. (A) The intracellular levels of amino acids between SCC vs. non-SCC cell lines. Each dot represents one amino acid. (B and C) Boxplots showing the levels of homocysteine and methionine in each cancer type. LC/MS data of (A–C) were collected from the CCLE database. (D and E) Box plots of indicated mRNA expression levels in UASCC from TCGA. (F) Box plots of LAT1 expression in published ESCC cohorts (G) Box plots of LAT1 expression in published HNSCC cohorts. (H) Kaplan–Meier analysis of TCGA HNSCC patient survival stratified by the mean level of LAT1 mRNA expression. (I and J) LC/MS for amino acid profiles upon knockdown of LAT1 in ESCC cell line (KYSE510).

Fig. 2.

TP63, KLF5, and SREBF1 directly coregulate LAT1 transcription by activating UASCC-specific enhancers and promoter. (A) IGV tracks of ChIP-Seq profiles of H3K27ac, TP63, KLF5, and SREBF1 at the LAT1 locus in indicated samples we published previously (10, 28, 29) (Upper) and H3K27ac Hi-ChIP contacts identified by a recent report (11) in various UASCC cell lines. (B) Enhancer and promoter activities measured by luciferase reporter assays after silencing of either TP63, KLF5, or SREBF1 in KYSE510 and KYSE150 cell lines. (C) The mRNA expression levels of TP63, KLF5, SREBF1, and LAT1 following knockdown of either TP63, KLF5, or SREBF1 in KYSE150 and KYSE510 cell lines. (D) Protein levels upon the knockdown of each transcription factor in the KYSE150 cell line. Tubulin was utilized as a loading control. All bar graphs were analyzed using three biological replicates. Mean ± SD is shown. P-values were determined by the two-tailed student t test. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 3.

LAT1 and methionine are indispensable for the survival and proliferation in UASCC cell lines and patient-derived tumor organoids. (A) mRNA expression of LAT1 after its knockdown in ESCC (KYSE150) and HNSCC (FADU) cell lines. (B) MTT assay for relative cell viability in ESCC and HNSCC cell lines upon knockdown of LAT1. (C) Quantification of colony growth in ESCC and HNSCC cell lines upon knockdown of LAT1. (D) Protein levels of LAT1, (E) number of colonies, and (F) cell viability of KYSE510 cells upon CRISPR/Cas9-mediated knockout of LAT1. One control clone and two independent knockout clones (C11 and D5) were measured. (G and H) Western blotting and colony formation following ectopic LAT1 over-expression in KYSE510 cell line. (I) Image of xenograft samples (I), (J) tumor size, and (K) weight from scramble (n = 8) and shLAT1 group (n = 8). (L) Cell viability upon restriction of methionine levels (3, 10, and 100 µM). (M) mRNA expression levels of LAT1 after its knockdown in patient-derived tumor organoids. (N) Representative images of organoids on day 1 and day 5. (O) Organoid viability measured by the WST1 assay at each time point. (P) Size distribution of organoids. All bar graphs were generated by biological replicates and shown as Mean ± SD. statistical significance was determined by two-tailed student’s t test. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 4.

LAT1 and EZH2 suppress a shared gene network. (A) Volcano plots showing enriched gene sets comparing two independent LAT1-knockdown groups vs. the control group. (B) GSEA line plots showing enrichment of the gene set of “LU_EZH2_TARGETS_UP” in siLAT1 vs. control groups. (C and D) GSEA line plots showing enrichment of up-regulated genes upon either LAT1-knockdown or EZH2-knockdown (two siRNAs per gene). (E) Heatmaps of relative mRNA expression levels of leading-edge genes upon silencing of either LAT1 (Left) or EZH2 (Right). (F) IGV tracks for three representative EZH2 binding genes in FADU and KYSE510 cell lines. (G) A pie chart summarizing EZH2 occupancy on the 17 leading-edge genes.

Fig. 5.

A LAT1-methionine-EZH2 axis. (A) Schematic diagram for LAT1-methionine-EZH2 cascade. (B) Relative mRNA expression levels of 17 leading-edge genes regulated by methionine restriction from the culture medium (3, 5, 10, and 100 μM). (C) Protein levels of LAT1 and H3K27me3 after LAT1 knockdown. (D) Protein levels of H3K27me3 regulated by methionine restriction from the culture medium. (E and F) H3K27me3 ChIP-qPCR for ZW10, FNBP1, and CASP7 gene promoters following either knockdown of LAT1 or deprivation of methionine levels in KYSE510 cell line. (G) Cell viability at indicated day point upon EZH2 knockdown. (H) Number of colonies following EZH2 knockdown for 2 wk in KYSE150 and FADU cell lines. All bar graphs were established using biological replicates and shown as Mean ± SD. P-value was determined by two-tailed student t test or two-way ANOVA test. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 6.

LAT1 and EZH2 inhibitors suppress the survival of UASCC cells and tumor organoids. (A) Representative images of organoids treated with Tazemetostat for 72 h. (B) Dose–response curves of three patient-derived tumor organoids (T1, T2, and T3) and normal organoids (N1 and N2) treated with Tazemetostat. (C) Representative images of organoids treated with JPH-203 for 72 h. (D) Dose–response curves of three patient-derived tumor organoids (T1, T2, and T3) and normal organoids (N1 and N2) treated with JPH-203 for 72 h (*P < 0.05; **P < 0.01; ***P < 0.001; vs. N1, and ^#P < 0.05; ^##P < 0.01; ^###P < 0.001; vs. N2). (E and F) Dose–response curves upon treatment with either Tazemetostat or JPH-203 in Scramble and two shLAT1 (-a and -b) stable cell lines for 72 h. Three biological replicates were shown as Mean ± SD. P-value was determined by two-tailed student t test. *P < 0.05; **P < 0.01; ***P < 0.001. (Scale bar, 20 μm.)

Fig. 7.

Methionine-restricted diet inhibits UASCC growth in vivo. (A) A schematic diagram of xenograft models for Scramble and LAT1-knockdown groups fed with either regular diet or methionine-restricted diet. (B) Tumor volumes for each group at indicated days (7, 11, 15, 19, and 24). (C and D) Image and weights of collected tumors from each group (n = 10). (E) Representative images of H&E staining and IHC staining of Ki-67, LAT1, and H3K27me3 for each group. (F) Relative mRNA expression levels of 17 genes in each group. Scale bar (H&E), 50 μm; Scale bar (IHC), 20 μm. All bar graphs are shown as Mean ± SD. P-value was determined by two-tailed student t test. *P < 0.05; **P < 0.01; ***P < 0.001