In a recent study published in Nature,Huang,Wong and their colleagues identified G2E3 as the major histone H3 lysine 14 mono-ubiquitin(H3K14ub)ligase in mammalian cells.1 By coupling G2E3-dependent H3K14 ubiquitination to the recruit-ment of SUV39H at pericentromeric chromatin,the authors define a conserved pathway that guides the reassembly of H3K9me3 domains after mitosis and secures the proper compartmentaliza-tion of chromatin.

Osteosarcoma is a highly heterogeneous and aggressive malignancy with a strong propensity for metastasis, highlighting the need to define its molecular drivers. Here, we report that ASB7 promotes tumor cell protrusion formation, invasion, migration, and lung metastasis. High expression of ASB7 is correlated with a poor prognosis. ASB7 forms an E3 ubiquitin ligase complex with CUL5 to ubiquitinate ATF2 at K383 and promote its proteasomal degradation. ATF2 reduction impairs HDAC6 recruitment to the ITGB2 promoter, thereby alleviating the transcriptional repression of ITGB2. Elevated ITGB2 expression subsequently promotes tumor lung metastasis. Our findings reveal that the ASB7-ATF2/HDAC6-ITGB2 axis regulates osteosarcoma metastasis and suggest potential treatment targets.

Histone H3 lysine 9 trimethylation (H3K9me3) is a defining mark of heterochromatin that silences transposable elements, stabilizes chromosome architecture, and guides developmental trajectories. Recent studies conceptualize H3K9me3 homeostasis as the interplay of three interconnected phases: initiation, propagation, and restriction. Initiation relies on sequence-specific and RNA-guided recruitment to seed histone methyltransferases at target loci. Propagation proceeds through self-reinforcing ‘read-write’ feedback but is constrained by chromatin barriers and regulated enzyme activity. During DNA replication, dilution of H3K9me3 by newly incorporated histones is counteracted by restoration guided by inherited parental nucleosomes, ensuring fidelity of heterochromatin transmission. This review summarizes recent advances in understanding H3K9me3 homeostasis, links its dysregulation to cancer, and highlights emerging therapeutic opportunities.

Aberrant transcriptional and epigenetic landscape plays crucial roles in the progression of bladder cancer (BC). However, effective therapeutic targets derived from these processes remain undeveloped. This study pinpoints SET-domain-containing protein 8 (SETD8) as a pivotal gene that promotes bladder tumor growth through a screening with a CRISPR-Cas9 library targeting transcriptional and epigenetic factors. BC patient samples display elevated SETD8 protein expression, and higher expression of SETD8 correlates with poorer prognosis. Further, MYC is identified as a novel substrate for SETD8. Specifically, SETD8 methylates MYC at lysine 412 (K412), disrupting the interaction between MYC and the E3 ubiquitin ligase CHIP, which results in MYC stabilization and ultimately promotes tumor growth both in vitro and in vivo. Moreover, this study uncovers that SUMOylation of SETD8 leads to SETD8 stabilization. The SUMOylated SETD8 further enhances MYC methylation and stabilization via SUMO-SIM interaction. Knocking down SETD8 or using the SETD8 specific inhibitor UNC0379 substantially reduces the protein level of MYC and inhibits the bladder tumor growth in vitro and in vivo. These findings provide strong support for the idea that targeting the SETD8/MYC axis offers a promising therapeutic approach for BC patient.

Osteosarcoma is the most common primary malignant bone cancer, characterized by a high incidence of lung metastasis and a lack of therapeutic targets. Here, by combining an in vivo CRISPR activation screen with the interactome of STUB1, a tumor suppressor in osteosarcoma, we identified that myeloid leukemia factor 2 (MLF2) promotes osteosarcoma metastasis. Mechanistically, MLF2 disrupted the interaction between BiP and IRE1α, thereby activating the IRE1α/XBP1-S-MMP9 axis. The E3 ligase STUB1 ubiquitinated MLF2 at Lys119 and targeted it for proteasomal degradation, whereas PIM3-mediated phosphorylation of MLF2 at Ser65 enhanced its stabilizing interaction with USP21. Our findings demonstrate that the PIM3/MLF2 axis is a critical regulator of osteosarcoma lung metastasis. We propose PIM3 as a potential therapeutic target for patients with osteosarcoma lung metastasis.

Tumor recurrence, metastasis, clinical drug resistance, and immune evasion are critical events in cancer progression, characterized by significant spatiotemporal heterogeneity and plasticity. Intercellular communication between tumor cells and other cells within the tumor microenvironment plays a pivotal role in these processes. Extracellular vesicles (EVs), heterogeneous secretory messengers carrying bioactive molecules, facilitate this cell-to-cell communication, thereby dynamically influencing cancer progression. Deciphering the mechanisms of EV formation and regulatory pathways and identifying key networks and targets in tumor metastasis, drug resistance, and immune response mediated by EVs will provide new insights into the understanding of cancer progression patterns and offer innovative strategies for cancer diagnosis and therapy.

Immune checkpoint blockade (ICB) therapies targeting the programmed cell death 1 (PD-1)/PD-1 ligand 1 (PD-L1) axis provide significant clinical benefits across multiple tumor types. Although interferon (IFN)-γ is essential for anti-tumor immunity, sustained IFN-γ signaling in the tumor microenvironment potently upregulates PD-L1 expression in tumor cells and induces profound T cell exhaustion, limiting the efficacy of ICB therapies. Therefore, further investigation into the regulation of IFN-γ-PD-L1 signaling is necessary for the development of more effective therapeutic strategies. Herein, transmembrane protein 199 (TMEM199) is identified as a novel regulator of IFN-γ-driven PD-L1 transcription. Mechanistically, TMEM199 and its important partner coiled-coil domain containing 115 (CCDC115) interact with IFNGR1/2 and facilitate their trafficking to RAB11A-positive recycling endosomes. TMEM199/CCDC115 also recruits transport protein particle (TRAPP) Ⅱ to the recycling endosomes and activates RAB11A, leading to enhanced IFNGR1/2 recycling and downstream PD-L1 upregulation. Collectively, these findings reveal that TMEM199 might be a promising therapeutic target for immunotherapy.

In eukaryotes, protein secretion plays essential roles in intercellular communications and extracellular niche-building. Protein secretion generally requires a signal sequence that targets cargos to the canonical secretory pathway consisting of the endoplasmic reticulum (ER), the Golgi apparatus, plasma membrane, and vesicles moving between these compartments. However, cytoplasmic proteins lacking signal sequences (e.g., IL1β, Acb1, FGF2) have been detected, and many have defined functions in the extracellular space, suggesting unconventional protein secretion (UcPS) via alternative pathways. In recent years, scientists have uncovered many new UcPS paradigms, reporting a plethora of mechanisms that collectively form a new field. The inaugural Cold Spring Harbor Asia (CSHA) conference on "Molecular Mechanisms and Physiology of Unconventional Secretion" is the first meeting to bring these researchers together, providing a collegial platform for information sharing at this exciting frontier of cell biology research.

Oncogenic mutations in EGFR often result in EGF-independent constitutive activation and aberrant trafficking and are associated with several human malignancies, including non-small cell lung cancer. A major consequence of EGFR mutations is the activation of the mechanistic target of rapamycin complex 1 (mTORC1), which requires EGFR kinase activity and downstream PI3K/AKT signaling, resulting in increased cell proliferation. However, recent studies have elucidated kinase-independent roles of EGFR in cell survival and cancer progression. Here, we report a cis mTORC1 activation function of EGFR that is independent of its kinase activity. Our results reveal that lysosomal localization of EGFR is critical to mTORC1 activation, where EGFR physically binds Rheb, acting as a guanine exchange factor (GEF) for Rheb, with its Glu804 serving as a potential glutamic finger. Genetic knock-in of EGFR-E804K in cells reduces the level of GTP-bound Rheb, and significantly suppresses mTORC1 activation, cell proliferation and tumor growth. Different tyrosine kinase inhibitors exhibit distinct effects on EGFR-induced mTORC1 activation, with afatinib, which additionally blocks EGFR’s GEF activity, causing a much greater suppression of mTORC1 activation and cell growth, and erlotinib, which targets only kinase activity, resulting in only a slight decrease. Moreover, a novel small molecule, BIEGi-1, was designed to target both the Rheb-GEF and kinase activities of EGFR, and shows a strong inhibitory effect on the viability of cells harboring EGFR mutants. These findings unveil a fundamental event in cell growth and suggest a promising strategy against cancers with EGFR mutations.

Cervical cancer metastasis is characterized by the systemic spread of tumor cells. However, the underlying mechanism remains incompletely understood. Herein, we demonstrate that RAB33A promoted metastasis by enhancing RhoC accumulation and that higher RAB33A expression predicted poorer prognosis in patients with cervical cancer. Mechanistically, RhoC typically degraded via canonical autophagy due to the binding of two LIR motifs (LC3 interaction region) in RhoC to LC3; however, RAB33A induced non-canonical autophagy, resulting in RhoC stabilization, which facilitated pseudopodia formation and consequently cervical cancer metastasis. The fusion of RAB33A-induced autophagosomes with lysosomes was impaired, as RAB33A inactivated RAB7 by interacting with TBC1D2A, a GTPase-activating protein that targets RAB7. Our findings reveal a pivotal role of the RAB33A-RhoC axis in cervical cancer metastasis, indicating that RhoC inhibitors may be beneficial for treating cervical cancer patients with high levels of RAB33A.

Anoikis is a critical obstacle to cancer metastasis. Colorectal cancer (CRC) exhibits a high rate of metastasis, leading to death, and the mechanisms involved in anoikis resistance are still unclear. We identified that the fatty acid oxidation (FAO) pathway was activated in detached CRC cells. Multiple genes in the FAO pathway, specifically the rate-limiting enzyme CPT1A, were upregulated in CRC cells grown in suspension. Reactive oxygen species elimination mediated by CPT1A in CRC cells was vital to anoikis resistance. In vivo experiments showed that CPT1A-suppressed CRC cells colonized the lung at a much lower rate than normal CRC cells, suggesting that CPT1A-mediated FAO activation increased metastatic capacity. In clinical tissue specimens from CRC patients, elevated expression of CPT1A was observed in metastatic sites compared with primary sites. Our results demonstrate that CPT1A-mediated FAO activation induces CRC cells to resist anoikis, suggesting that CPT1A is an attractive target for treating metastatic CRC.

The maintenance of histone H3 lysine 9 trimethylation (H3K9me3) involves the recognition of preexisting modifications by heterochromatin protein 1 (HP1), which recruits the methyltransferase suppressor of variegation 3-9 homolog 1 (SUV39H1) to methylate the adjacent newly incorporated histones, establishing a positive feedback loop. However, how this positive feedback is restricted to maintain H3K9me3 homeostasis remains largely unknown. We performed an unbiased genome-scale CRISPR-Cas9 screen and identified CUL5ASB7 E3 ubiquitin ligase as a negative regulator of H3K9me3. ASB7 is recruited to heterochromatin by HP1 and promotes SUV39H1 degradation. During mitosis, cyclin-dependent kinase 1 (CDK1) phosphorylates ASB7, preventing its interaction with SUV39H1, leading to SUV39H1 stabilization and H3K9me3 restoration. Our findings reveal a dynamic circuit involving HP1, SUV39H1, and ASB7 that governs H3K9me3 homeostasis, ensuring faithful epigenetic inheritance and preventing excessive heterochromatin formation.

Rafeesome, a newly identified multivesicular body (MVB)-like organelle, forms through the fusion of RAB22A-mediated ER-derived noncanonical autophagosomes with RAB22A-positive early endosomes. However, the mechanism underlying the formation of RAB22A-mediated noncanonical autophagosomes remains unclear. Herein, we report a secretory ER-phagy pathway in which the assembly of RAB22A/TMEM33/RTN4 induces the clustering of high-molecular-weight RTN4 oligomers, leading to ER membrane remodeling. This remodeling drives the biogenesis of ER-derived RTN4-positive noncanonical autophagosomes, which are ultimately secreted as TMEM33-marked RAB22A-induced extracellular vesicles (R-EVs) via Rafeesome. Specifically, RAB22A interacts with the tubular ER membrane protein TMEM33, which binds to the TM2 domain of the ER-shaping protein RTN4, promoting RTN4 homo-oligomerization and thereby generating RTN4-enriched microdomains. Consequently, the RTN4 microdomains may induce high curvature of the ER, facilitating the bud scission of RTN4-positive vesicles. These vesicles are transported by ATG9A and develop into isolation membranes (IMs), which are then anchored by LC3-II, a process catalyzed by the ATG12-ATG5-ATG16L1 complex, allowing them to grow into sealed RTN4 noncanonical autophagosome. While being packaged into these ER-derived intermediate compartments, ER cargoes bypass lysosomal degradation and are directed to secretory autophagy via the Rafeesome-R-EV route. Our findings reveal a secretory ER-phagy pathway initiated by the assembly of RAB22A/TMEM33/RTN4, providing new insights into the connection between ER-phagy and extracellular vesicles.

Bladder cancer (BC) is one of the most common tumors characterized by a high rate of relapse and a lack of targeted therapy. Here, YEATS domain-containing protein 4 (YEATS4) is an essential gene for BC cell viability using CRISPR-Cas9 library screening is reported, and that HUWE1 is an E3 ligase responsible for YEATS4 ubiquitination and proteasomal degradation by the Protein Stability Regulators Screening Assay. KAT8-mediated acetylation of YEATS4 impaired its interaction with HUWE1 and consequently prevented its ubiquitination and degradation. The protein levels of YEATS4 and KAT8 are positively correlated and high levels of these two proteins are associated with poor overall survival in BC patients. Importantly, suppression of YEATS4 acetylation with the KAT8 inhibitor MG149 decreased YEATS4 acetylation, reduced cell viability, and sensitized BC cells to cisplatin treatment. The findings reveal a critical role of the KAT8/YEATS4 axis in both tumor growth and cisplatin sensitivity in BC cells, potentially generating a novel therapeutic strategy for BC patients.

Intratumor heterogeneity is one of the major features of cancers, leading to aggressive disease and treatment failure. Cancer stem-like cells (CSCs) are believed to give rise to the heterogeneous cell types within tumors. Hence, understanding the regulatory mechanism underlying the recurrence process of heterogeneous tumor by CSCs could facilitate the development of CSC-targeted therapies. Here, utilizing single-cell transcriptomics, we present the molecular profile of osteosarcoma CSCs-derived heterogeneous tumors consisting of CSC clusters, osteoprogenitor and differentiated cell types, such as pre-osteoblasts, osteoblasts and chondroblasts. Furthermore, by constructing the comprehensive map of modulated genes during CSCs self-renewal and differentiation, we identify RAN exhibiting specific peak expression in osteosarcoma CSCs clusters which is transcriptionally up-regulated by MYBL2. Functionality, MYBL2-RAN pathway promotes the CSCs self-renewal by enhancing the nuclear accumulation of MYC protein, which in turn boosts the overexpression of RAN as a positive feedback. Importantly, blockage of MYBL2-RAN pathway sensitizes CSCs to cisplatin treatment and synergistically enhanced the cisplatin-induced cytotoxicity. Both MYBL2 and RAN are highly expressed in clinical osteosarcoma tissues which indicate poor prognosis. Collectively, our study provides advanced insights into the regeneration process of heterogeneous tumor originating from CSCs and highlights the MYBL2-RAN pathway as a promising target for CSC-based therapy in osteosarcoma.