STK11/LKB1 Loss of Function Is Associated with Global DNA Hypomethylation and S-Adenosyl-Methionine Depletion in Human Lung Adenocarcinoma.

STK11 (liver kinase B1, LKB1) is the fourth most frequently mutated gene in lung adenocarcinoma, with loss of function observed in up to 30% of all cases. Our previous work identified a 16-gene signature for LKB1 loss of function through mutational and nonmutational mechanisms. In this study, we applied this genetic signature to The Cancer Genome Atlas (TCGA) lung adenocarcinoma samples and discovered a novel association between LKB1 loss and widespread DNA demethylation. LKB1-deficient tumors showed depletion of S-adenosyl-methionine (SAM-e), which is the primary substrate for DNMT1 activity. Lower methylation following LKB1 loss involved repetitive elements (RE) and altered RE transcription, as well as decreased sensitivity to azacytidine. Demethylated CpGs were enriched for FOXA family consensus binding sites, and nuclear expression, localization, and turnover of FOXA was dependent upon LKB1. Overall, these findings demonstrate that a large number of lung adenocarcinomas exhibit global hypomethylation driven by LKB1 loss, which has implications for both epigenetic therapy and immunotherapy in these cancers. SIGNIFICANCE: Lung adenocarcinomas with LKB1 loss demonstrate global genomic hypomethylation associated with depletion of SAM-e, reduced expression of DNMT1, and increased transcription of repetitive elements.

Cytoplasmic mRNA recapping has limited impact on proteome complexity.

The m7G cap marks the 5' end of all eukaryotic mRNAs, but there are also capped ends that map downstream within spliced exons. A portion of the mRNA transcriptome undergoes a cyclical process of decapping and recapping, termed cap homeostasis, which impacts the translation and stability of these mRNAs. Blocking cytoplasmic capping results in the appearance of uncapped 5' ends at native cap sites but also near downstream cap sites. If translation initiates at these sites the products would lack the expected N-terminal sequences, raising the possibility of a link between mRNA recapping and proteome complexity. We performed a shotgun proteomics analysis on cells carrying an inducible inhibitor of cytoplasmic capping. A total of 21 875 tryptic peptides corresponding to 3565 proteins were identified in induced and uninduced cells. Of these, only 29 proteins significantly increased, and 28 proteins significantly decreased, when cytoplasmic capping was inhibited, indicating mRNA recapping has little overall impact on protein expression. In addition, overall peptide coverage per protein did not change significantly when cytoplasmic capping was inhibited. Together with previous work, our findings indicate cap homeostasis functions primarily in gating mRNAs between translating and non-translating states, and not as a source of proteome complexity.

RNA-binding proteins and heat-shock protein 90 are constituents of the cytoplasmic capping enzyme interactome.

The N7-methylguanosine cap is added in the nucleus early in gene transcription and is a defining feature of eukaryotic mRNAs. Mammalian cells also possess cytoplasmic machinery for restoring the cap at uncapped or partially degraded RNA 5' ends. Central to both pathways is capping enzyme (CE) (RNA guanylyltransferase and 5'-phosphatase (RNGTT)), a bifunctional, nuclear and cytoplasmic enzyme. CE is recruited to the cytoplasmic capping complex by binding of a C-terminal proline-rich sequence to the third Src homology 3 (SH3) domain of NCK adapter protein 1 (NCK1). To gain broader insight into the cellular context of cytoplasmic recapping, here we identified the protein interactome of cytoplasmic CE in human U2OS cells through two complementary approaches: chemical cross-linking and recovery with cytoplasmic CE and protein screening with proximity-dependent biotin identification (BioID). This strategy unexpectedly identified 66 proteins, 52 of which are RNA-binding proteins. We found that CE interacts with several of these proteins independently of RNA, mediated by sequences within its N-terminal triphosphatase domain, and we present a model describing how CE-binding proteins may function in defining recapping targets. This analysis also revealed that CE is a client protein of heat shock protein 90 (HSP90). Nuclear and cytoplasmic CEs were exquisitely sensitive to inhibition of HSP90, with both forms declining significantly following treatment with each of several HSP90 inhibitors. Importantly, steady-state levels of capped mRNAs decreased in cells treated with the HSP90 inhibitor geldanamycin, raising the possibility that the cytotoxic effect of these drugs may partially be due to a general reduction in translatable mRNAs.