A miR-99a-TGF-β1 feedback circuit drives miR-99a upregulation.

EV-miR-99a targets NLRP2 and activates NF-kB signaling in fibroblasts.

The tumor microenvironment is crucial for cancer progression, but the mechanisms underlying the tumor-immune cell interactions in it remain poorly understood. Here, we identified latent transforming growth factor-β (TGFβ) binding protein 4 (LTBP4) deficiency in colorectal cancer (CRC) as a critical driver that reprogrammed tumor-associated macrophages (TAMs) and induced a distinct subset, which promoted tumor progression by coordinating immune evasion and extracellular matrix (ECM) remodeling. Clinically, LTBP4 deficiency correlated with CRC progression and poor patient survival. Ltbp4 knockout markedly promoted tumor growth and metastasis in immunocompetent mice, an effect attenuated in immunodeficient hosts, establishing the essential role of host immunity in mediating the effects of LTBP4 deficiency. Single-cell RNA sequencing revealed that LTBP4 deficiency induced a mannose receptor C-type 1-positive (MRC1+)/CD44+ TAM subset and correlated with reduced CD8+ T cell infiltration. Mechanistically, LTBP4 deficiency increased active TGFβ1 levels, which acted in a paracrine manner to up-regulate MRC1 in TAMs, whereas autocrine signaling induced HAS2 (hyaluronan synthase 2) expression and hyaluronan production to increase CD44. CD44 signaling in TAMs up-regulated matrix metalloproteinases for collagen degradation, whereas MRC1 mediated collagen internalization, cooperatively remodeling the ECM to facilitate tumor invasion. The TGFβ1-driven MRC1+/CD44+ TAMs further suppressed CD8+ T cell function by diminishing the C-X-C motif chemokine ligand 16-C-X-C motif chemokine receptor 6 (CXCL16-CXCR6) axis. Therapeutically, targeted depleting MRC1+/CD44+ TAMs enhanced the efficacy of PD-1 (programmed cell death-1) blockade in LTBP4-deficient tumors. Our study positions LTBP4 as a key modulator of tumor progression and reveals a therapeutic strategy for LTBP4-deficient CRC.

miR-99a associates with CRC progression and prognosis.

EVs from highly metastatic CRC cells promote fibroblast activation.

The association between folate metabolism abnormalities and the development of colorectal cancer (CRC) remains controversial. Here, we report that the folate exerts a tumor-suppressive role in CRC; however, the manifestation of this effect is restricted by the expression level of folate transporter SLC46A1 in CRC cells. Multi-cohort profiling revealed significant downregulation of SLC46A1 in CRC tissues compared to adjacent normal tissues, where low expression independently predicted poor overall survival. Functional studies demonstrated that SLC46A1-mediated folate uptake suppressed tumor proliferation, migration, and invasion both in vitro and in vivo. Mechanistically, SLC46A1 deficiency restricted intracellular folate availability and impaired cellular methylation potential, as evidenced by a reduced SAM/SAH ratio, leading to DNA hypomethylation at specific sites such as the FOS proto-oncogene promoter. This epigenetic reprogramming triggers transcriptional activation of key oncogenic effectors CCND1, BCL2, and PLAU involved in CRC progression. Clinically, we found a significant inverse correlation between SLC46A1 expression and folate levels in tumor interstitial fluids of CRC, suggesting impaired folate uptake in low SLC46A1 tumors. Multi-color immunofluorescence across two cohorts further demonstrated conserved inverse associations between SLC46A1 and FOS expression in primary tumors and metastatic lesions. This study elucidates the molecular mechanism by which folate inhibits CRC progression through the “SLC46A1-epigenetic-transcriptional regulation” axis, providing mechanistic insights into folate deficiency-driven CRC progression and biomarkers for precision CRC intervention.

Dysfunctional tumor vasculature limits immune checkpoint blockade efficacy. We have recently established that endothelial SPEN deficiency promotes tumor vessel normalization by suppressing RNA polymerase I (RNAPI)-mediated rDNA transcription via inducing disrupted ribosome biogenesis and nucleolar stress, but whether this pathway could be practically employed in immunotherapy has been unclear. In the current study, analysis of publicly available single‑cell RNA‑seq datasets revealed that endothelial SPEN expression was downregulated in clinical responders, including colorectal cancer patients achieving pathological complete response (pCR) after PD‑1 blockade and lung adenocarcinoma patients showing major pathological response (MPR) after neoadjuvant PD‑1 blockade plus chemotherapy. Using endothelial-specific SPEN knockout mice (eSPEN-/-), we demonstrate that SPEN loss in established tumors remodels the immune microenvironment by enhancing the infiltration and perivascular accumulation of both CD8+ and CD4+ T-cells, and in addition, boosting the effector function of CD8+ T-cells. A combined therapy of anti-PD-1 with CX-5461, a small-molecule inhibitor of RNAPI yielded better tumor inhibition than monotherapy with anti-PD-1 alone. This was attributable to increased T-cell infiltration and a marked expansion of polyfunctional CD8⁺ T-cells capable of producing both IFN-γ and TNF-α. In summary, our study identifies endothelial RNAPI, which is regulated by SPEN, as a target of normalizing tumor-immune microenvironment and that the RNAPI inhibitor, which is under clinical trial, is potentially useful to improve PD-1 blocker in cancer therapy.

Safety assessment of LY2584702 in mice.

Metabolic dysfunction-associated steatotic liver disease (MASLD) and its progressive inflammatory form, metabolic dysfunction-associated steatohepatitis (MASH), constitute major causes of hepatic morbidity worldwide. Central to the pathophysiology of these conditions is the progressive impairment of liver sinusoidal endothelial cells (LSECs), whose characteristic fenestrated architecture is indispensable for hepatic homeostasis. Capillarization, defined as the loss of LSEC fenestrations, propels disease progression by disrupting the equilibrium between pro-fenestration and pro-capillarization signaling cascades. This review critically appraises the LSEC Fenestration Signaling Nexus as a central, targetable mechanism in metabolic liver disease and assesses emerging therapeutic strategies engineered to restore fenestrations. We present a translational roadmap that prioritizes combinatorial pharmacological approaches and LSEC-specific biomarkers for precision medicine. This integrative framework furnishes new perspectives for rational drug development and individualized therapeutic strategies in metabolic liver disease.

Inflammation and epithelial-to-mesenchymal transition are hallmarks of cancer progression. A better understanding of the mechanisms driving these processes could help uncover strategies to treat and prevent metastasis. In this study, we found that extracellular vesicle (EV)-mediated cross-talk between colorectal cancer cells and fibroblasts facilitates inflammation and promotes metastasis. Fibroblasts were highly activated in primary tumors from patients with colorectal cancer with metastatic disease, and EVs secreted from highly metastatic colorectal cancer cells promoted fibroblast activation. Mechanistically, EV-packaged miR-99a-5p (EV-miR-99a) specifically targeted NLRP2 mRNA in fibroblasts and activated the proinflammatory NFκB signaling pathway, thereby converting normal fibroblasts into cancer-associated fibroblasts (CAF). EV-miR-99a-activated CAFs enhanced the migratory capacity of colorectal cancer cells by secreting CCL7, which potently induced epithelial-to-mesenchymal transition by increasing the expression of multiple E-cadherin repressors via the CCR5-mTOR-p70S6K pathway. Expression of miR-99a in colorectal cancer cells was upregulated by TGFβ1 secreted from CAFs in an NFκB-dependent manner, forming an miR-99a/TGFβ1 regulatory circuit. The communication between colorectal cancer cells and fibroblasts engendered a proinflammatory niche that facilitated metastasis, which could be abolished by treatment with the p70S6K inhibitor LY2584702. Clinically, EV-miR-99a levels in the plasma correlated with the metastatic status of patients with colorectal cancer. Together, these findings highlight the metastasis-promoting function of an inflammatory fibroblast niche induced by cancer cell-derived EVs and provide potential targets for the prediction and management of colorectal cancer metastasis. SIGNIFICANCE:Extracellular vesicle-mediated cross-talk between colorectal cancer cells and fibroblasts orchestrates a proinflammatory niche that induces and facilitates metastasis and provides potential targets for disease prediction and therapeutic intervention.

Overactivation of Notch signaling in liver sinusoidal endothelial cells (LSECs) is an established driver of hepatic fibrosis, yet its precise upstream regulators and downstream effectors remain poorly defined, limiting therapeutic translation. Here, we demonstrate that FBXW7, the primary E3 ubiquitin ligase responsible for NOTCH1 degradation, serves as a critical suppressor of liver fibrosis. Using endothelial-specific Fbxw7 knockout mice subjected to three distinct fibrosis models, we show that FBXW7 loss exacerbates liver fibrosis, promotes LSEC capillarization, and enhances hepatic stellate cell activation. Transcriptomic analysis identified Sema3g as the top upregulated gene in FBXW7‑deficient LSECs, and conditioned medium from these cells stimulated HSC activation through SEMA3G‑dependent paracrine signaling. Moreover, nanoparticle-mediated delivery of Sema3g-targeting siRNA specifically to LSECs substantially ameliorated injury-induced liver fibrosis in Fbxw7 knockout mice. Mechanistically, FBXW7 loss stabilizes NOTCH1, leading to enhanced Sema3g transcription. Consistent with these findings, human cirrhotic samples and murine fibrotic livers exhibit reduced FBXW7 expression alongside elevated active NOTCH1 and SEMA3G in LSECs. Collectively, our results demonstrate that FBXW7 mitigates liver fibrosis by degrading NOTCH1 to transcriptionally silence SEMA3G, and suggest that targeting the FBXW7/NOTCH1/SEMA3G axis represents a promising therapeutic strategy for fibrotic liver disease. The authors reported a randomized trial showing that transcutaneous auricular vagus nerve stimulation reduces postoperative liver injury in patients undergoing partial hepatectomy.

B-cell acute lymphoblastic leukemia (B-ALL) is a hematological malignancy characterized by the aberrant accumulation of malignant and immature B cells in the bone marrow. Recent reports including ours have demonstrated that RNA modifications play pivotal roles in B-ALL progression and drug resistance. In the current study, we show that fat mass and obesity-associated protein (FTO), a demethylase of N6-methyladenosine (m6A) RNA modifications, is highly expressed in relapsed or refractory (R/R) B-ALL patients and B-ALL cell lines. In human B-ALL cells, FTO knockdown inhibited proliferation and cell cycle progression in vitro, while FTO overexpression exhibited opposite effects. Moreover, FTO knockdown significantly attenuated tumorigenesis in vivo after transplantation into immune-compromised mice as shown by reduced tumor burden and extended mouse survival. Interestingly, our research suggested that FTO overexpression resulted in altered cytoplasmic and mitochondrial ribosome biogenesis, and FTO knockdown led to a nucleolar stress-like morphologic change and mitochondrial dysfunction in B-ALL cells. Mechanistically, we found that FTO upregulated the expression of a group of ribosomal proteins via m6A-modification, among which RPS15a, RPL9, MRPS16 and MRPL44 were the most prominent ones and confirmed at the mRNA and protein levels. FTO upregulates RPS15a, RPL9, MRPS16 and MRPL44 by mitigating YTHDF2-mediated m6A-mRNA decay. Comparatively, although FTO knockdown induced B-ALL cell apoptosis mildly, it synergized with Doxorubicin to promote apparent B-ALL cell death. Furthermore, we found that the FTO inhibitor FB23-2 combined with Doxorubicin markedly repressed B-ALL progression in vivo, accompanied by nucleolar stress-like changes and mitochondrial dysfunction. In summary, our data suggest that FTO is a critical RNA epigenetic promotor of B-ALL, and targeted FTO blockade synergizing with Doxorubicin could be a potential therapy for B-ALL, likely by inhibiting cytoplasmic and mitochondrial ribosome biogenesis.

Abnormal infiltration and activation of monocyte-derived macrophages (moMFs) contribute significantly to thoracic aortic dissection (TAD). The transcription factor RBPJ mediates canonical Notch signaling and modulates macrophage activation, but the role and mechanism of RBPJ in macrophages in TAD remains unclear. Here, we show that RBPJ was upregulated in macrophages infiltrating the aorta in TAD patients and BAPN-induced mouse model. Myeloid-specific Rbpj ablation protected mice from TAD, reducing death, aortic damage, macrophage infiltration, and M1-like polarization while enhancing M2-like polarization. Because moMFs dominate aorta as shown in public scRNA-seq data, and RBPJ is upregulated in moMFs compared with blood monocytes/macrophages, we assumed that mechanical force, specifically cyclic stretch, might be one of the environmental cues of macrophage activation in aorta. Indeed, bone marrow-derived macrophages (BMDMs) loaded with cyclic stretch upregulated RBPJ expression in a Piezo1-dependent way, accompanied by increased M1-like polarization, and Rbpj ablation cancelled the force-induced M1-like polarization. By RNA-sequencing, we found that cyclic stretch induced a metabolic reprogram of BMDMs characterized by upregulation of glycolysis-related genes and HIF1α, which was dependent on RBPJ. Further analyses showed that cyclic stretch upregulated PDK1, a negative regulator of pyruvate dehydrogenase (PDH), which was abrogated by RBPJ deficiency. Based on these findings, we administered dichloroacetate (DCA), a pan-PDK inhibitor, in TAD mice, and found that DCA significantly attenuated BAPN-induced TAD in mice. Therefore, our results demonstrate that RBPJ is required for pro-inflammatory moMFs activation, likely by mediating mechanotransduction-induced glycolysis via PDK1 upregulation, and PDK1 inhibitors such as DCA are potential therapeutics for TAD.

Background: The chaotic, over-activated tumor vasculature promotes tumor growth and erodes most current therapies. Although Notch activation critically regulates angiogenesis, the broad roles of Notch has dampened its druggability. Methods: Gene-modified mice with a Cdh5-CreERT transgene were employed to activate/block Notch signaling in endothelial cells (ECs). Multiple transcriptome analyses were conducted to compare gene expression profiles. qRT-PCR and western blotting were used to determine gene expression level. Immunofluorescence and flow cytometry were used to observe morphological alterations and immune microenvironment in tumors. Nanoparticles (PEI-PEG-cRGD) were used to deliver siRNA into tumor ECs (TECs) in vivo. Results: Genetic Notch activation or blockade in TECs normalizes or deteriorates tumor vessels, respectively. Single-cell RNA sequencing showed that Notch activation selectively reduced the proliferating TEC subset, which accounted for about 30% of TECs and gave rise to other TEC subsets. Notch activation or blockade downregulated or upregulated MYC, respectively. MYC overexpression canceled Notch activation-induced proliferation arrest of TECs in vitro, and a MYC inhibitor normalized tumor vessels in RBPj deficient mice, suggesting that MYC is the authentic Notch target in normalizing tumor vessels. Nanoparticles encapsulated with MYC siRNA (EC-siMYC) or miR-218 (EC-miR-218), a Notch-downstream miRNA suppressing MYC, were able to mitigate Notch inhibition-induced tumor vessel defects. Combination of cisplatin with MYC blockade exhibited improved therapeutic effects. Moreover, MYC blockade promoted T cell infiltration and enhanced anti-PD1 immunotherapy. Conclusions: Together, our data have demonstrated that Notch activation normalizes tumor vessels by repressing the proliferating TEC subset via MYC, and targeting endothelial MYC using nanoparticles bearing siRNA or miRNA is an efficient strategy for tumor anti-angiogenic therapy.

AIMS:Angiogenesis, a tightly regulated process involving dynamic endothelial cell (EC) proliferation, is critical in both physiological and pathological contexts such as ocular neovascular disorders. While Notch signaling is known to regulate angiogenesis, its downstream molecular mechanisms remain incompletely understood. MAIN METHODS:Gene-modified mice with a Cdh5-CreERT transgene were generated to block Notch signaling in ECs. Primary HUVECs were cultured in vitro. Gene expression were analyzed via qRT-PCR, western blotting, and immunofluorescence. Transcriptional regulation was investigated using reporter and ChIP assays. EC proliferation and migration were assessed through EdU incorporation, Transwell, and wound healing assays, respectively. Angiogenesis was evaluated in vivo using Matrigel plug, retinal angiogenesis, oxygen-induced retinopathy (OIR), and choroidal neovascularization (CNV) models. KEY FINDING:Notch activation upregulated, whereas Notch blockade downregulated EZH2 expression in both mRNA and protein levels. Mechanistically, Notch signaling directly suppresses EZH2 promoter activity, thereby transcriptionally repressing EZH2 expression. Functionally, EZH2 inhibition impaired EC proliferation and sprouting angiogenesis, while EZH2 overexpression enhanced these processes. Furthermore, EZH2 inhibition reversed the pro-angiogenic effects of Notch blockade. At the molecular level, EZH2 stabilized MYC protein by modulating Thr58 phosphorylation and regulating MYC stability factors, thereby preventing its proteasomal degradation. MYC overexpression rescued the angiogenesis defects caused by EZH2 inhibition. Importantly, EZH2 was upregulated in OIR and CNV models, and pharmacological EZH2 inhibition (GSK126) effectively suppressed pathological angiogenesis. SIGNIFICANCE:Our findings not only elucidate the novel role of EZH2 in mediating Notch function in angiogenesis but also provide a promising therapeutic strategy for treating neovascularization-related diseases with EZH2 inhibitors.

Hepatocellular carcinoma (HCC) is the most common form of primary liver cancer and poses a significant global health challenge. In recent years, tRNA-derived small RNAs (tsRNAs) have gained significant attention due to their potential role in various cancers, including HCC. In this study, we reported that tRF-34-P4R8YP9LON4VHM expression was elevated in HCC tissues and cell lines. The association between tRF-34-P4R8YP9LON4VHM expression and HCC patients' clinicopathological parameters was determined using tissue microarrays of 90 patients, and we found that it was positively associated with the level of AFP, tumour size, microvascular density (MVD), and TNM stage. We performed CCK8, colony formation assay, EdU, cell cycle analysis, transwell assay, and tube formation assay to verify that tRF-34-P4R8YP9LON4VHM could enhance HCC proliferation, migration, invasion, and tumour cell-induced angiogenesis in vitro and in vivo. Mechanistically, tRF-34-P4R8YP9LON4VHM could downregulate DAB2IP expression by directly targeting its 3'-UTR, consequently activating the MEK/ERK signalling pathway and promoting the secretion of VEGFA from HCC cells into the supernatant. In conclusion, our research indicated that tRF-34-P4R8YP9LON4VHM might act as a crucial player in molecular mechanisms and provide novel treatment strategies for HCC patients.

Background The effectiveness of immune checkpoint inhibitors in colorectal cancer (CRC) is limited due to the low tumor neoantigen load and low immune infiltration in most microsatellite-stable (MSS) tumors. This study aimed to develop a mitochondria-targeted photodynamic therapy (PDT) approach to provoke host antitumor immunity of MSS-CRC and elucidate the underlying molecular mechanisms.Methods The role and mechanism of mitochondria-targeted PDT in inhibiting CRC progression and inducing pyroptosis were evaluated both in vitro and in vivo. The immune effects of PDT sensitization on PD-1 blockade were also assessed in CT26 and 4T1 tumor-bearing mouse models.Results Here, we report that PDT using IR700DX-6T, a photosensitizer targeting the mitochondrial translocation protein, may trigger an antitumor immune response initiated by pyroptosis in CRC. Mechanistically, IR700DX-6T-PDT produced reactive oxygen species on light irradiation and promoted downstream p38 phosphorylation and active caspase3 (CASP3)-mediated cleavage of gasdermin E (GSDME), subsequently inducing pyroptosis. Furthermore, IR700DX-6T-PDT enhanced the sensitivity of MSS-CRC cells to PD-1 blockade. Decitabine, a demethylation drug used to treat hematologic neoplasms, disrupted the abnormal methylation pattern of GSDME in tumor cells, enhanced the efficacy of IR700DX-6T-PDT, and elicited a potent antitumor immune response in combination with PD-1 blockade and IR700DX-6T-PDT.Conclusion Our work provides clear a understanding of immunogenic cell death triggered by mitochondria-targeted PDT, offering a new approach for enhancing the efficacy of PD-1 blockade in CRC.

Taurine is used to bolster immunity, but its effects on antitumor immunity are unclear. Here, we report that cancer-related taurine consumption causes T cell exhaustion and tumor progression. The taurine transporter SLC6A6 is correlated with aggressiveness and poor outcomes in multiple cancers. SLC6A6-mediated taurine uptake promotes the malignant behaviors of tumor cells but also increases the survival and effector function of CD8+ T cells. Tumor cells outcompete CD8+ T cells for taurine by overexpressing SLC6A6, which induces T cell death and malfunction, thereby fueling tumor progression. Mechanistically, taurine deficiency in CD8+ T cells increases ER stress, promoting ATF4 transcription in a PERK-JAK1-STAT3 signaling-dependent manner. Increased ATF4 transactivates multiple immune checkpoint genes and induces T cell exhaustion. In gastric cancer, we identify a chemotherapy-induced SP1-SLC6A6 regulatory axis. Our findings suggest that tumoral-SLC6A6-mediated taurine deficiency promotes immune evasion and that taurine supplementation reinvigorates exhausted CD8+ T cells and increases the efficacy of cancer therapies.