Nuclear KRT19 is a transcriptional corepressor promoting histone deacetylation and liver tumorigenesis.

BACKGROUND AND AIMS: Epigenetic reprogramming and escape from terminal differentiation are poorly understood enabling characteristics of liver cancer. Keratin 19 (KRT19), classically known to form the intermediate filament cytoskeleton, is a marker of stemness and worse prognosis in liver cancer. This study aimed to address the functional roles of KRT19 in liver tumorigenesis and to elucidate the underlying mechanisms. APPROACH AND RESULTS: Using multiplexed genome editing of hepatocytes in vivo, we demonstrated that KRT19 promoted liver tumorigenesis in mice. Cell fractionation revealed a previously unrecognized nuclear fraction of KRT19. Tandem affinity purification identified histone deacetylase 1 and REST corepressor 1, components of the corepressor of RE-1 silencing transcription factor (CoREST) complex as KRT19-interacting proteins. KRT19 knockout markedly enhanced histone acetylation levels. Mechanistically, KRT19 promotes CoREST complex formation by enhancing histone deacetylase 1 and REST corepressor 1 interaction, thus increasing the deacetylase activity. ChIP-seq revealed hepatocyte-specific genes, such as hepatocyte nuclear factor 4 alpha ( HNF4A ), as direct targets of KRT19-CoREST. In addition, we identified forkhead box P4 as a direct activator of aberrant KRT19 expression in liver cancer. Furthermore, treatment of primary liver tumors and patient-derived xenografts in mice suggest that KRT19 expression has the potential to predict response to histone deacetylase 1 inhibitors especially in combination with lenvatinib. CONCLUSIONS: Our data show that nuclear KRT19 acts as a transcriptional corepressor through promoting the deacetylase activity of the CoREST complex, resulting in dedifferentiation of liver cancer. These findings reveal a previously unrecognized function of KRT19 in directly shaping the epigenetic landscape in cancer.

New progress in roles of TGF-ß signaling crosstalks in cellular functions, immunity and diseases.

The family of secreted dimeric proteins known as the Transforming Growth Factor-ß (TGF-ß) family plays a critical role in facilitating intercellular communication within multicellular animals. A recent symposium on TGF-ß Biology - Signaling, Development, and Diseases, held on December 19-21, 2023, in Hangzhou, China, showcased some latest advances in our understanding TGF-ß biology and also served as an important forum for scientific collaboration and exchange of ideas. More than twenty presentations and discussions at the symposium delved into the intricate mechanisms of TGF-ß superfamily signaling pathways, their roles in normal development and immunity, and the pathological conditions associated with pathway dysregulation.

SNX8 enables lysosome reformation and reverses lysosomal storage disorder.

Lysosomal Storage Disorders (LSDs), which share common phenotypes, including enlarged lysosomes and defective lysosomal storage, are caused by mutations in lysosome-related genes. Although gene therapies and enzyme replacement therapies have been explored, there are currently no effective routine therapies against LSDs. During lysosome reformation, which occurs when the functional lysosome pool is reduced, lysosomal lipids and proteins are recycled to restore lysosome functions. Here we report that the sorting nexin protein SNX8 promotes lysosome tubulation, a process that is required for lysosome reformation, and that loss of SNX8 leads to phenotypes characteristic of LSDs in human cells. SNX8 overexpression rescued features of LSDs in cells, and AAV-based delivery of SNX8 to the brain rescued LSD phenotypes in mice. Importantly, by screening a natural compound library, we identified three small molecules that enhanced SNX8-lysosome binding and reversed LSD phenotypes in human cells and in mice. Altogether, our results provide a potential solution for the treatment of LSDs.

IRF3 activates RB to authorize cGAS-STING-induced senescence and mitigate liver fibrosis.

Cytosolic double-stranded DNA surveillance by cyclic GMP-AMP synthase (cGAS)-Stimulator of Interferon Genes (STING) signaling triggers cellular senescence, autophagy, biased mRNA translation, and interferon-mediated immune responses. However, detailed mechanisms and physiological relevance of STING-induced senescence are not fully understood. Here, we unexpectedly found that interferon regulatory factor 3 (IRF3), activated during innate DNA sensing, forms substantial endogenous complexes in the nucleus with retinoblastoma (RB), a key cell cycle regulator. The IRF3-RB interaction attenuates cyclin-dependent kinase 4/6 (CDK4/6)-mediated RB hyperphosphorylation that mobilizes RB to deactivate E2 family (E2F) transcription factors, thereby driving cells into senescence. STING-IRF3-RB signaling plays a notable role in hepatic stellate cells (HSCs) within various murine models, pushing activated HSCs toward senescence. Accordingly, IRF3 global knockout or conditional deletion in HSCs aggravated liver fibrosis, a process mitigated by the CDK4/6 inhibitor. These findings underscore a straightforward yet vital mechanism of cGAS-STING signaling in inducing cellular senescence and unveil its unexpected biology in limiting liver fibrosis.

A chaperone-like function of FUS ensures TAZ condensate dynamics and transcriptional activation.

The Hippo pathway has important roles in organ development, tissue homeostasis and tumour growth. Its downstream effector TAZ is a transcriptional coactivator that promotes target gene expression through the formation of biomolecular condensates. However, the mechanisms that regulate the biophysical properties of TAZ condensates to enable Hippo signalling are not well understood. Here using chemical crosslinking combined with an unbiased proteomics approach, we show that FUS associates with TAZ condensates and exerts a chaperone-like effect to maintain their proper liquidity and robust transcriptional activity. Mechanistically, the low complexity sequence domain of FUS targets the coiled-coil domain of TAZ in a phosphorylation-regulated manner, which ensures the liquidity and dynamicity of TAZ condensates. In cells lacking FUS, TAZ condensates transition into gel-like or solid-like assembles with immobilized TAZ, which leads to reduced expression of target genes and inhibition of pro-tumorigenic activity. Thus, our findings identify a chaperone-like function of FUS in Hippo regulation and demonstrate that appropriate biophysical properties of transcriptional condensates are essential for gene activation.

Smad4 sequestered in SFPQ condensates prevents TGF-ß tumor-suppressive signaling.

Loss of TGF-ß growth-inhibitory responses is a hallmark of human cancer. However, the molecular mechanisms underlying the TGF-ß resistance of cancer cells remain to be fully elucidated. Splicing factor proline- and glutamine-rich (SFPQ) is a prion-like RNA-binding protein that is frequently upregulated in human cancers. In this study, we identified SFPQ as a potent suppressor of TGF-ß signaling. The ability of SFPQ to suppress TGF-ß responses depends on its prion-like domain (PrLD) that drives liquid-liquid phase separation (LLPS). Mechanistically, SFPQ physically restrained Smad4 in its condensates, which excluded Smad4 from the Smad complex and chromatin occupancy and thus functionally dampened Smad-dependent transcriptional responses. Accordingly, SFPQ deficiency or loss of phase separation activities rendered human cells hypersensitive to TGF-ß responses. Together, our data identify an important function of SFPQ through LLPS that suppresses Smad transcriptional activation and TGF-ß tumor-suppressive activity.

Innate immune sensing of lysosomal dysfunction drives multiple lysosomal storage disorders.

Lysosomal storage disorders (LSDs), which are characterized by genetic and metabolic lysosomal dysfunctions, constitute over 60 degenerative diseases with considerable health and economic burdens. However, the mechanisms driving the progressive death of functional cells due to lysosomal defects remain incompletely understood, and broad-spectrum therapeutics against LSDs are lacking. Here, we found that various gene abnormalities that cause LSDs, including Hexb, Gla, Npc1, Ctsd and Gba, all shared mutual properties to robustly autoactivate neuron-intrinsic cGAS-STING signalling, driving neuronal death and disease progression. This signalling was triggered by excessive cytoplasmic congregation of the dsDNA and DNA sensor cGAS in neurons. Genetic ablation of cGAS or STING, digestion of neuronal cytosolic dsDNA by DNase, and repair of neuronal lysosomal dysfunction alleviated symptoms of Sandhoff disease, Fabry disease and Niemann-Pick disease, with substantially reduced neuronal loss. We therefore identify a ubiquitous mechanism mediating the pathogenesis of a variety of LSDs, unveil an inherent connection between lysosomal defects and innate immunity, and suggest a uniform strategy for curing LSDs.

Loss of ZNRF3/RNF43 Unleashes EGFR in Cancer.

ZNRF3 and RNF43 are closely related transmembrane E3 ubiquitin ligases with significant roles in development and cancer. Conventionally, their biological functions have been associated with regulating WNT signaling receptor ubiquitination and degradation. However, our proteogenomic studies have revealed EGFR as the most negatively correlated protein with ZNRF3/RNF43 mRNA levels in multiple human cancers. Through biochemical investigations, we demonstrate that ZNRF3/RNF43 interact with EGFR via their extracellular domains, leading to EGFR ubiquitination and subsequent degradation facilitated by the E3 ligase RING domain. Overexpression of ZNRF3 reduces EGFR levels and suppresses cancer cell growth in vitro and in vivo, whereas knockout of ZNRF3/RNF43 stimulates cell growth and tumorigenesis through upregulated EGFR signaling. Together, these data highlight ZNRF3 and RNF43 as novel E3 ubiquitin ligases of EGFR and establish the inactivation of ZNRF3/RNF43 as a driver of increased EGFR signaling, ultimately promoting cancer progression. This discovery establishes a connection between two fundamental signaling pathways, EGFR and WNT, at the level of cytoplasmic membrane receptor, uncovering a novel mechanism underlying the frequent co-activation of EGFR and WNT signaling in development and cancer.

The RNF214-TEAD-YAP signaling axis promotes hepatocellular carcinoma progression via TEAD ubiquitylation.

RNF214 is an understudied ubiquitin ligase with little knowledge of its biological functions or protein substrates. Here we show that the TEAD transcription factors in the Hippo pathway are substrates of RNF214. RNF214 induces non-proteolytic ubiquitylation at a conserved lysine residue of TEADs, enhances interactions between TEADs and YAP, and promotes transactivation of the downstream genes of the Hippo signaling. Moreover, YAP and TAZ could bind polyubiquitin chains, implying the underlying mechanisms by which RNF214 regulates the Hippo pathway. Furthermore, RNF214 is overexpressed in hepatocellular carcinoma (HCC) and inversely correlates with differentiation status and patient survival. Consistently, RNF214 promotes tumor cell proliferation, migration, and invasion, and HCC tumorigenesis in mice. Collectively, our data reveal RNF214 as a critical component in the Hippo pathway by forming a signaling axis of RNF214-TEAD-YAP and suggest that RNF214 is an oncogene of HCC and could be a potential drug target of HCC therapy.