The intrinsic ability of cancer cells to evade death underpins tumorigenesis, progression, metastasis, and the survival of drug-tolerant persister (DTP) cells. Herein, we discovered that the small GTPase ARF6 plays a central role in tumor survival by fortifying RAF oncoprotein levels. ARF6 activation was sufficient to increase BRAFV600E, ARAF, and CRAF proteins through a post-transcriptional mechanism, while sustained inhibition of ARF6 eventually led to decay. In a genetically engineered model of aggressive melanoma, tumor-specific Arf6 deletion attenuated BRAFV600E protein expression and MAPK signaling and prevented rapid tumor progression. In human melanoma cells, pharmacologic inhibitors of BRAFV600E uniformly induced swift activation of ARF6, driving a positive feedback loop that restored MAPK-driven anti-apoptotic signaling and supported drug-tolerant survival and growth. Furthermore, in patient-derived melanoma xenografts with innate or clinically acquired resistance to MAPK inhibitors, ARF6 silencing alone significantly suppressed tumor growth in vivo. When combined with BRAF and MEK targeted therapy in vitro, inhibition of ARF6 markedly reduced survival and drug-tolerant growth. Collectively, these findings reveal a previously unknown mechanism of maintaining BRAFV600E protein expression that preserves the MAPK pathway during targeted therapy. This ARF6-dependent mechanism may be exploited in BRAFV600E driven cancers as a therapeutic vulnerability.

Colorectal cancer (CRC) is one of the most commonly diagnosed and globally spread malignant diseases. Cancer-associated fibroblasts (CAFs) are key architects of the tumor microenvironment, yet their origin, stability, and interconvertibility remain poorly understood. Using transcriptomic profiling of fibroblasts from colorectal cancer (CRC) patients, we identify highly expressed (HEX) markers that define fibroblast subpopulations and uncover mechanisms governing their plasticity. We find that ADH1B marks normal colon-associated fibroblasts (NAFs), which consist of PI16-NAFs and ADAMDEC1-NAFs. ITGA3 delineates the total CAF population, which comprises myofibroblastic CAFs (myCAFs), whose characterizing markers were associated with poor prognosis and proteolytic inflammatory CAFs (piCAFs), characterized by markers not associated with prognosis. An AGT/TGM2-expressing fibroblast subset is present in both healthy and tumor tissues, suggesting alternative trajectories to the classical NAF-to-CAF transition model. While PI16-NAFs, AGT/TGM2-fibroblasts, and myCAFs maintain stable identities in long-term culture, the ADAMDEC1-NAF and piCAF phenotypes are lost in vitro. ITGA3-CAFs demonstrate dynamic plasticity, with TGF-β stably inducing myCAF formation and TNF-α or inhibition of DNA methylation promoting transient piCAF emergence. These findings redefine fibroblast heterogeneity in CRC and reveal a coexisting stable and plastic fibroblast network that may be amenable to modulation and provides a framework for future functional and translational studies. We identified highly expressed markers (HEX markers) to distinguish CAFs, NAFs and corresponding subpopulations in CRC. ADH1B characterized NAFs, which consisted of stable (solid outline) PI16-NAFs and unstable (dashed outline) ADAMDEC1-NAFs. ITGA3 identified CAFs consisting of stable myCAFs associated with poor prognosis and unstable piCAFs not associated with prognosis. AGT/TGM2 fibroblasts did not express ADH1B or ITGA3, were stable in culture and could be detected in both healthy colon and CRC. Treatment of PI16-NAFs with LPS or IFN-γ induced ADAMDEC1-NAFs, TGF-β the formation of myCAFs, while treatment with TNF-α led to the formation of piCAFs. Reduced DNA methylation converted myCAFs and PI16-NAFs into piCAFs.

Hepatocellular carcinoma (HCC) is a prevalent and aggressive malignancy, notorious for its high recurrence, substantial drug resistance, and poor prognosis. Although METTL1, a key regulator of RNA m7G modification, is implicated in the progression of various cancers, its role and underlying mechanisms in HCC remain poorly understood. This study identified that METTL1 was significantly upregulated in HCC tissues, correlating with advanced stages and poor survival. Knockdown of METTL1 inhibited cell proliferation, migration, and invasion, which was reversed by restoring METTL1 expression. Multi-omics analysis indicated that METTL1 regulated gene expression through m7G modification, particularly in the Wnt, mTOR signaling pathways, and amino acid metabolism (especially asparagine metabolism). Further analysis revealed that METTL1 increased the stability and upregulated the expression levels of asparagine synthetase (ASNS) mRNA through m7G modification, thereby reprogramming asparagine metabolism and activating the mTOR pathway, ultimately promoting HCC progression. In conclusion, METTL1 regulated ASNS mRNA stability and expression via m7G modification, driving HCC malignancy through reprogramming of asparagine metabolism and activation of the mTOR signaling pathway.

Immunotherapy targeting the programmed cell death-1 (PD-1)/programmed cell death-ligand 1 (PD-L1) (PD-1/PD-L1) axis has revolutionized cancer treatment, yet its efficacy in hepatocellular carcinoma (HCC) remains limited. Emerging evidence suggests that gut microbiota plays a pivotal role in modulating tumor immune microenvironments (TIME), offering a novel avenue to enhance immunotherapy outcomes. This study investigates the regulatory effects of Lactobacillus kefiranofaciens (LK) on the TIME in HCC, focusing on its modulation of Suppressor of cytokine signaling 3 (SOCS3) expression and the Janus-activated kinase-signal transducer and activator of transcription (JAK-STAT) signaling pathway, with the goal of improving responses to anti-PD-1/PD-L1 therapy. LK was isolated from kefir grains and identified through genomic sequencing. In vitro assays, including Cell Counting Kit-8 (CCK-8), 5-Ethynyl -2'- deoxyuridine (EdU) staining, colony formation, Transwell, and apoptosis detection, were conducted using Hepa1-6 HCC cells. In vivo, subcutaneous and orthotopic HCC mouse models were treated with LK to assess tumor progression. Single-cell RNA sequencing (scRNA-seq) and Bulk RNA-seq analyses were performed to identify key signaling pathways and therapeutic targets. SOCS3 expression was manipulated via lentiviral transfection to validate its role in immunotherapy enhancement. LK significantly inhibited HCC cell growth, migration, and invasion, while promoting apoptosis in vitro. In vivo, LK treatment reduced tumor size and improved immune cell infiltration, particularly T cells and NK cells. Transcriptomic analysis revealed that LK upregulates SOCS3, suppresses the JAK-STAT signaling pathway, and reduces PD-L1 expression, enhancing T cell-mediated immune responses. This study highlights the potential of gut microbiota modulation, specifically through LK, to enhance the efficacy of anti-PD-1/PD-L1 immunotherapy in HCC by targeting SOCS3 and the JAK-STAT pathway. These findings provide a new therapeutic approach for improving immunotherapy outcomes in HCC. Gut probiotics modulate the immune microenvironment to enhance the response of liver cancer patients to anti-PD-1/PD-L1 immunotherapy: molecular mechanisms.

Ecotropic viral integration site 1 (EVI1) is essential for hematopoietic stem cell maintenance, and its aberrant expression is a significant adverse prognostic indicator in myeloid leukemia. EVI1 overexpression typically occurs due to chromosomal rearrangement involving 3q26. However, aberrant EVI1 expression is still observed in numerous cases without 3q26 abnormalities, leading to similarly poor outcomes, while the mechanism behind EVI1 overexpression in these cases remains largely unknown. Here, we performed genome-wide CRISPR screening using cells with GFP knock-in at the EVI1 locus and identified zinc finger protein 91 (ZFP91) was the leading activator of EVI1. ZFP91 knockout significantly reduced EVI1 expression and cell proliferation. We also showed that ZFP91 binds to the EVI1 promoter, enhancing H3K4me3/H3K27ac and chromatin accessibility. Our data showed that the ZFP91-EVI1 axis plays a critical role for activation of EVI1 in myeloid leukemia. Our screening approach represents a powerful and unbiased method for identifying expression regulators that can be broadly applied across a range of contexts.

The AAA+ ATPase VPS4 drives the ESCRT machinery in diverse intracellular membrane remodeling events, including endocytic receptor sorting, membrane repair, and autophagosome closure. Tumor cells often lose one VPS4 paralog (VPS4A or VPS4B), making them dependent on the remaining enzyme and creating a potential therapeutic vulnerability. Inhibiting VPS4 induces cancer cell-autonomous death and may also modulate the immune microenvironment, although the underlying mechanisms remain unclear. Here, we report that VPS4 inhibition triggered upregulation of cytokine and innate immune signaling, along with canonical NF-κB, stress response, and cell death pathways in murine rhabdomyosarcoma (RMS) cells. Pharmacological and genetic analyses identified the cGAS-STING-TBK1-IRF3 axis, activated by cytoplasmic mitochondrial DNA, as the primary driver of cytokine induction. In an orthotopic syngeneic RMS model, VPS4 inhibition suppressed tumor growth while fostering a more immunogenic microenvironment. Although STING was dispensable for VPS4 inhibition-induced RMS cell death, its loss reduced natural killer and dendritic cell infiltration and attenuated the overall anti-tumor effects of VPS4 inhibition. These findings establish a dual role for VPS4 inhibition in inducing tumor cell death and promoting anti-tumor immunity, highlighting the therapeutic potential of targeting VPS4 vulnerability in cancer.

The Hippo pathway is an evolutionarily conserved signaling cascade whose dysregulation is implicated in a wide range of diseases. While many RNA-binding proteins (RBPs) regulate this pathway through canonical functions such as modulating mRNA stability and translation, the potential for RBP-mediated regulation via non-canonical, RNA-binding-independent mechanisms remains poorly defined. Here, we report that the RBP TIAL1 exhibits oncogenic properties in hepatocellular carcinoma, promoting cancer cell proliferation, migration, and invasion. Mechanistically, TIAL1 directly interacts with the core Hippo component SAV1, disrupting the MST1-SAV1 interaction and thereby suppressing Hippo signaling and activating YAP. Notably, this regulatory function is independent of the RNA-binding activity of TIAL1. Furthermore, extracellular stimuli such as energy surplus and EGF significantly upregulate TIAL1 expression, linking microenvironmental cues to Hippo pathway dysregulation. Together, our results reveal a previously unrecognized, RNA-binding-independent mode of RBP-mediated regulation, in which TIAL1 serves as a molecular integrator that conveys extracellular signals to the Hippo pathway to drive hepatocellular carcinoma progression, providing potential avenues for therapeutic intervention.

Aberrantly enhanced DNA damage repair contributes to therapy resistance and poor prognosis in nasopharyngeal carcinoma (NPC), but its regulatory mechanisms remain unclear. Stress granules (SGs) mediate tumor stress adaptation, yet their role in NPC DNA damage repair is unknown. Here, we show that SGs are significantly enriched in NPC cells under stress, and the SG core protein G3BP1 is highly expressed in NPC tissues (n = 111), correlating with metastasis and poor survival. Mechanistically, under stress, N-acetyltransferase 10 (NAT10)-catalyzed N4-acetylcytosine (ac4C) modification targets mRNAs of DNA repair genes (ATF3, LIG1, RNF168) to SGs, protecting them from degradation. Upon stress relief, these mRNAs are released for translation, enhancing DNA damage repair. The G3BP1/NAT10/ATF3 axis is critical for NPC DNA repair and metastasis, as blocking this axis (via G3BP1 depletion, NAT10 inhibitor remodelin, or ATF3 knockout) inhibits tumor growth and metastasis in vitro and in vivo. This study uncovers a novel ac4C-dependent mechanism by which SGs regulate DNA damage repair in NPC, identifying the G3BP1/NAT10/ATF3 axis as a potential therapeutic target for improving NPC prognosis.

Radioresistance remains the primary cause of radiotherapy failure in non-small cell lung cancer (NSCLC). This study investigated the regulatory role of HMOX1-mediated ferroptosis in NSCLC radiosensitivity. Radioresistant cell models (H1650R/H1975R) were established through fractionated irradiation of parental H1650/H1975 cells. Transcriptomic analysis by RNA sequencing revealed significant HMOX1 suppression in resistant cells. Functional validation demonstrated that HMOX1 overexpression enhanced radiation sensitivity via ferroptosis induction, whereas HMOX1 knockdown aggravated radioresistance. Mechanistic investigations identified USP7 as a key deubiquitinating enzyme that stabilizes KEAP1 through K48-linked polyubiquitin chain cleavage, thereby promoting NRF2 ubiquitination and suppressing HMOX1 transcription. Pharmacological inhibition using KI696 blocked KEAP1-NRF2 interaction, restoring HMOX1 expression. Notably, the USP7 inhibitor GNE-6640 destabilized KEAP1, upregulated NRF2/HMOX1 axis activity, and triggered ferroptosis in resistant cells. In vivo studies confirmed that GNE-6640 synergized with radiotherapy to suppress tumor growth and pulmonary metastasis in xenograft and NSG mouse models, as monitored by bioluminescence imaging. These findings establish the USP7-KEAP1-NRF2-HMOX1 axis as a critical determinant of radioresistance, demonstrating that targeted USP7 inhibition with GNE-6640 reactivates ferroptosis and restores radiosensitivity. This dual-mechanistic approach provides a novel therapeutic strategy to overcome treatment resistance in NSCLC.

PIWI proteins, a subfamily of the PAZ-PIWI domain (PPD) protein family, are traditionally regarded as germline factors that partner with PIWI-interacting RNAs (piRNAs) to silence transposons and regulate gene expression. However, growing evidence implicates PIWI proteins as oncogenic drivers in diverse somatic cancers, often acting through piRNA-independent mechanisms that remain incompletely understood. Here, we integrate transcriptomic, translatomic, and proteomic profiling of wild-type versus PIWIL1-knockout gastric cancer cells to uncover a non-canonical, translational role for PIWIL1, one of the four human PIWI proteins. We find that PIWIL1 selectively enhances the translation of 5'-terminal oligopyrimidine (TOP) mRNAs by activating mTOR complex 1 (mTORC1). Mechanistically, PIWIL1 interacts with the R2TP chaperone complex (RUVBL1-RUVBL2-RPAP3-PIH1D1) and promotes its association with TELO2, facilitating mTOR-RAPTOR assembly and mTORC1 activation. Functionally, PIWIL1 deficiency sensitizes gastric cancer cells to mTOR inhibition, and in clinical samples, PIWIL1 expression positively correlates with mTORC1 pathway activity. Together, these findings define a novel piRNA-independent mechanism through which PIWIL1 contributes to tumor progression, extend PIWI-mediated translational control from the germline to human cancers, and establish PIWIL1 as a potential therapeutic target for gastric cancer in synergy with mTOR inhibition.