Introduction: Head and neck squamous cell carcinoma (HNSCC) development and progression is accompanied by pervasive metabolic dysregulations. In this study, we aim to characterize metabolic features in HNSCC and their functional significance in cancer biology.

Results: We conducted pan-cancer metabolic analyses and unexpectedly observed that methionine cycle is particularly high in HNSCC samples. Specifically, across all cancer types, HNSCC showed the highest levels of methionine and homocysteine. Moreover, noted that the methionine transporter, SLC7A5, exhibited a highly HNSCC-specific expression pattern in pan-cancer data. Importantly, higher expression of SLC7A5 was significantly associated with worse survival of patients in HNSCC.

We next performed SLC7A5 loss-of-function experiments by either siRNAs or CRISPR/Cas9 genome editing, finding that depletion of SLC7A5 suppressed significantly proliferation and increased apoptosis of HNSCC cells. Ectopic SLC7A5 over-expression promoted colony formation of HNSCC cells. Moreover, SLC7A5-silencing markedly inhibited mouse xenograft growth in vivo. Consistently, methionine deprivation from culture media decreased cell viability and colony formation. Importantly, the effect of methionine deprivation was dependent on SLC7A5, since SLC7A5-depletion largely rescued phenotypes caused by methionine deprivation.

We next probed possible mechanisms underlying the notable HNSCC-specific expression pattern of SLC7A5. Chromatin Immunoprecipitation Sequencing (ChIP-seq) and chromosome conformation assays (HiChIP) identified a strong super-enhancer of SLC7A5 in HNSCC cells. Using luciferase reported assays, we further showed that a recently established HNSCC-specific core regulatory circuitry TP63/KLF5/SREBF1 trio-occupied this super-enhancer and activated the transcription of SLC7A5 in HNSCC cells in a cell-type-specific manner.

We next performed transcriptomic sequencing upon knockdown of SLC7A5 by independent siRNAs. Notably, the most significantly enriched gene-set was an EZH2 gene signature. Intrigued by this observation, we further performed RNA-seq upon knockdown of EZH2 in HNSCC cells, which reproduced the strong enrichments aforementioned. Furthermore, knockdown of EZH2 inhibited the proliferation and viability of HNSCC cells, consistent with the phonotypical changes triggered by silencing of SLC7A5 or methionine deprivation.

As an well-recognized cancer driver, EZH2 is a central histone methyltransferase catalyzing the addition of methyl groups to histone H3, leading to epigenetic silencing. Importantly, EZH2 activity critically depends on the intracellular methionine level. Given the striking similarity between EZH2- and SLC7A5-regualted transcriptomes, we hypothesized that SLC7A5-mediated uptake of methionine could promote EZH2 activity, regulating the epigenome of HNSCC cells. We thus performed metabolic profiling using mass spectrometry and confirmed that intracellular methionine was the most downregulated amino acid following SLC7A5-knockdown. Importantly, EZH2 ChIP-seq and rescue assays showed that EZH2 genome occupancy and methyltransferase activity relied on the levels of both SLC7A5 protein and methionine.

Conclusions: This study identifies a prominent HNSCC-specific amino acid transporter, SLC7A5, whose expression is driven by HNSCC core regulatory circuitry TP63/KLF5/SREBF1 through a super-enhancer. Overexpression of SLC7A5 protein leads to a uniquely high methionine cycle in HNSCC, which activates EZH2 methyltransferase, de-regulates the HNSCC epigenome and promotes tumor aggressiveness. These findings establish an important mechanistic crosstalk between metabolic and epigenomic reprogramming, as well as its biological significance in cancer biology. Moreover, this novel SLC7A5-methionine-EZH2 cascade is pharmacologically actionable and may represent a promising drug target against HNSCC.