Recent studies have indicated that the expression of ubiquitin-specific protease 51 (USP51), a novel deubiquitinating enzyme (DUB) that mediates protein degradation as part of the ubiquitin‒proteasome system (UPS), is associated with tumor progression and therapeutic resistance in multiple malignancies. However, the underlying mechanisms and signaling networks involved in USP51-mediated regulation of malignant phenotypes remain largely unknown. The present study provides evidence of USP51's functions as the prominent DUB in chemoresistant triple-negative breast cancer (TNBC) cells. At the molecular level, ectopic expression of USP51 stabilized the 78 kDa Glucose-Regulated Protein (GRP78) protein through deubiquitination, thereby increasing its expression and localization on the cell surface. Furthermore, the upregulation of cell surface GRP78 increased the activity of ATP binding cassette subfamily B member 1 (ABCB1), the main efflux pump of doxorubicin (DOX), ultimately decreasing its accumulation in TNBC cells and promoting the development of drug resistance both in vitro and in vivo. Clinically, we found significant correlations among USP51, GRP78, and ABCB1 expression in TNBC patients with chemoresistance. Elevated USP51, GRP78, and ABCB1 levels were also strongly associated with a poor patient prognosis. Importantly, we revealed an alternative intervention for specific pharmacological targeting of USP51 for TNBC cell chemosensitization. In conclusion, these findings collectively indicate that the USP51/GRP78/ABCB1 network is a key contributor to the malignant progression and chemotherapeutic resistance of TNBC cells, underscoring the pivotal role of USP51 as a novel therapeutic target for cancer management.

Cyclin-dependent kinase 9 (CDK9) is a member of the transcription CDK subfamily and plays a role in transcriptional regulation. Selective CDK9 degraders possess potent clinical advantages over reversible CDK9 inhibitors. Herein, we report the first ATG101-recruiting selective CDK9 degrader, AZ-9, based on the hydrophobic tag kinesin degradation technology. AZ-9 showed significant degradation effects and selectivity toward other homologous cell cycle CDKs in vitro and in vivo, which could also affect downstream related phenotypes. Mechanism research revealed that AZ-9 recruits ATG101 to initiate the autophagy–lysosome pathway, and forms autophagosomes through the recruitment of LC3, which then fuses with lysosomes to degrade CDK9 and the partner protein Cyclin T1. These dates validated the existence of non-proteasomal degradation pathway of hydrophobic driven protein degradation strategy for the first time, which might provide research ideas for chemical induction intervention on other types of pathogenic proteins.

Arginine methylation is a critical post-translational modification that plays multifaceted biological functions. However, the manipulation of protein arginine methylation largely depends on genetic or pharmaceutic inhibition of the regulatory enzymes, protein arginine methyltransferases (PRMTs), or non-methylation substitution of corresponding arginine residue to lysine or alanine of protein of interest (POI), which inevitably affects other substrates, or disrupts the structure of POI. Thus, it urges an approach to specifically modulate the arginine methylation of a POI under physiological conditions. To this end, we report the discovery of a methylation tagging system (MeTAG), that enables targeted modification of protein arginine methylation. Through bridging the methyltransferase PRMT5 proximity to a POI, MeTAG facilitates the arginine methylation of POIs, including known arginine methylated proteins, androgen receptor (AR) and protein kinase B (AKT), as well as a neo-substrate E1A binding protein (p300), in a reversible and PRMT5-dependent manner. Moreover, MeTAG can regulate downstream signaling in a methylation dependent manner, leading to downregulation of PSMA mRNA level and activation of AKT. Therefore, MeTAG represents a feasible approach to modulate protein methylation and thereby perturbs protein function in biological and therapeutic contexts.

Cuproptosis, a recently identified form of regulated cell death triggered by excess intracellular copper, has emerged as a promising cytotoxic strategy for cancer therapy. However, the therapeutic efficacy of copper ionophores such as elesclomol (ES) is often hindered by cellular copper homeostasis mechanisms that limit copper influx and cuproptosis induction. To address this challenge, we developed a nanoagent utilizing outer membrane vesicle (OMV) derived from Akkermansia muciniphila (Akk) for co-delivery of antioxidant 1 copper chaperone (Atox1)-targeting siRNA and ES (siAtox1/ES@OMV) to tumors. In vitro, we demonstrated that Atox1 knockdown via siRNA significantly disrupted copper export mechanisms, resulting in elevated intracellular copper levels. Simultaneously, ES facilitated efficient copper influx and mitochondrial transport, leading to Fe–S cluster depletion, increased proteotoxic stress, and robust cuproptosis. In vivo, siAtox1/ES@OMV achieved targeted tumor delivery and induced pronounced cuproptosis. Furthermore, leveraging the immunomodulatory properties of OMVs, siAtox1/ES@OMV promoted T-cell infiltration and the activation of tumor-reactive cytotoxic T cells, enhancing tumor immune responses. The combination of siAtox1/ES-induced cuproptosis and immunogenic cell death synergistically suppressed tumor growth in both subcutaneous breast cancer and orthotopic rectal cancer mouse models. This study highlights the potential of integrating copper homeostasis disruption with a copper ionophore using an immunomodulatory OMV-based vector, offering a promising combinatorial strategy for cancer therapy.

Sonodynamic therapy (SDT) can potentially induce immunogenic cell death in tumor cells, leading to the release of ATP, and facilitating the initiation of an immune response. Nevertheless, the enzymes CD39 and CD73 can swiftly convert ATP into immunosuppressive adenosine (ADO), resulting in an immunosuppressive tumor microenvironment (TME). This study introduced a nanomedicine (QD/POM1@NP@M) engineered to reprogram TME by modulating the CD39/CD73/ADO pathway. The nanomedicine encapsulated sonosensitizers silver sulfide quantum dots, and the CD39 inhibitor POM1, while also incorporating homologous tumor cell membranes to enhance targeting capabilities. This integrated approach, on the one hand, stimulates the release of ATP via SDT, thereby initiating the immune response. In addition, it reduced the accumulation of ADO by inhibiting CD39 activity, which ameliorated the immunosuppressive TME. Upon administration, the nanomedicine demonstrated substantial anti-tumor efficacy by facilitating the infiltration of anti-tumor immune cells, while reducing the immunosuppressive cells. This modulation effectively transformed the TME from an immunologically “cold” state to a “hot” state. Furthermore, combined with the checkpoint inhibitor α-PDL1, the nanomedicine augmented systemic anti-tumor immunity and promoted the establishment of long-term immune memory. This study provides an innovative strategy for combining non-invasive SDT and ATP-driven immunotherapy, offering new ideas for future cancer treatment.

The αPD-L1 antibody-based immune checkpoint blockade therapy is still limited by the poor clinical response rate as it is mainly utilized to block surface PD-L1 on tumor cells while ignoring abundant PD-L1 exosomes secreted in the environment, causing tumor immune evasion. Here, we proposed an exosome biogenesis inhibition strategy to suppress tumor exosomes secretion from the source, reducing the inhibitory effect on T cells and enhancing chemo-immunotherapy efficacy. We developed sulfafurazole homodimers (SAS) with disulfide linkages, effectively releasing the drug in response to glutathione (GSH) and inhibiting 4T1 tumor-derived exosomes secretion. Subsequently, gemcitabine (Gem) was encapsulated to induce immunogenic cell death (ICD). Consequently, Gem@SAS inhibited the secretion of tumor exosomes by more than 70%, increased proliferation and granzyme B secretion ability of T cells by more than 2 times, and showed superior efficacy in breast cancer treatment as well as lung metastasis of breast cancer.

Obesity usually exacerbates the immunosuppressive tumor microenvironment (ITME), hindering CD8⁺ T cell infiltration and function, which further represents a significant barrier to the efficacy of immunotherapy. Herein, a multifunctional liposomal system (CR-Lip) for encapsulating celastrol (CEL) was utilized to remodel obesity-related ITME and improve cancer immunotherapy, wherein Ginsenoside Rg3 (Rg3) was detected interspersed in the phospholipid bilayer and its glycosyl exposed on the surface of the liposome. CR-Lip had a relatively uniform size (116.5 nm), facilitating favorable tumor tissue accumulation through the interaction between Rg3 and glucose transporter 1 overexpressed in obese tumor cells. Upon reaching the tumor region, CR-Lip was found to induce the immunogenic cell death (ICD) of HFD tumor cells. Notably, the level of PHD3 in HFD tumor cells was effectively boosted by CR-Lip to effectively block metabolic reprogramming and increase the availability of major free fatty acids fuel sources. In vivo, experiments studies revealed that the easy-obtained nano platform stimulated enhanced the production of various cytokines in tumor tissues, DC maturation, CD8⁺ T-cell infiltration, and synergistic anticancer therapeutic potency with aPD-1 (tumor inhibition rate = 82.1%) towards obesity-related melanoma. Consequently, this study presented an efficacious approach to tumor immunotherapy in obese mice by encompassing tumor eradication, inducing ICD, and reprogramming metabolism. Furthermore, it offered a unique insight into a valuable attempt at the immunotherapy of obesity-associated related tumors.

Lacking therapeutic targets highlights the crucial roles of chemotherapy and radiotherapy in the clinical management of triple-negative breast cancer (TNBC). To relieve the side effects of the chemoradiotherapy combination regimen, we design and develop a self-assembled micelle nanosystem consisting of perfluorocarbon chain-modified cisplatin prodrug. By incorporating perfluorodecalin, this nanosystem can effectively carry ozone and promote irradiation-derived reactive oxygen species (ROS) production. By leveraging the perfluorocarbon sidechain, the nanosystem exhibits efficient internalization by TNBC cells and effectively escapes from lysosomal entrapment. Under X-ray irradiation, ozone-generated ROS disrupts the intracellular redox balance, thereby facilitating the release of cisplatin in a reduction-responsive manner mediated by reduced glutathione. Moreover, oxygen derived from ozone decomposition enhances the efficacy of radiotherapy by alleviating tumor hypoxia. Notably, the combination of irradiation with ozone-loaded cisplatin prodrug nano system synergistically prompts antitumor efficacy and reduces cellular/systemic toxicity in vitro and in vivo. Furthermore, the combo regimen remodels the tumor microenvironment into an immune-favored state by triggering immunogenic cell death and relieving hypoxia, which provides a promising foundation for a combination regimen of immunotherapy. In conclusion, our nanosystem presents a novel strategy for integrating chemotherapy and radiotherapy to optimize the efficacy and safety of TNBC clinical treatment.

Single-cell analysis of phenotypic plasticity could improve the development of more effective therapeutics. Still, the development of tools to measure single-cell heterogeneity has lagged due to difficulties in manipulating and culturing single cells. Here, we describe a single-cell culture and phenotyping platform that employs a starburst microfluidic network and automatic liquid handling system to capture single cells for long-term culture and multi-dimensional analysis and quantify their clonal properties via their surface biomarker and secreted cytokine/growth factor profiles. Studies performed on this platform found that cells derived from single-cell cultures maintained phenotypic equilibria similar to their parental populations. Single-cell cultures exposed to chemotherapeutic drugs stochastically disrupted this balance to favor stem-like cells. They had enhanced expression of mRNAs and secreted factors associated with cell signaling, survival, and differentiation. This single-cell analysis approach can be extended to analyze more complex phenotypes and screen responses to therapeutic targets.

Organic anion-transporting polypeptides (OATP) transporter function, which mediates many drugs' liver uptake, was investigated as a molecular determinant of pharmacokinetic variability. Whole-body PET imaging using ¹¹C-glyburide, a metabolically stable OATP probe, was performed in 16 healthy humans. Ten subjects underwent another ¹¹C-glyburide PET acquisition after OATP inhibition using rifampicin. Subjects were sorted according to age and sex: males<30y (24.0 ± 3.2 y, n = 7), males>50y (57.5 ± 5.6 y, n = 4), and females>50y (60.6 ± 2.4 y, n = 5). The blood-to-liver transfer rate (k_uptake) was estimated to describe OATP function. Rifampicin decreased k_uptake (−73 ± 13%, P < 0.001) and liver exposure (−50 ± 10%, P < 0.001) while increasing exposure in blood (+24 ± 24%, P < 0.01), myocardium, spleen, and brain (P < 0.05). No evidence of extra-hepatic rifampicin-inhibitable transport of ¹¹C-glyburide was found. Baseline liver exposure was 42.6 ± 18.4% higher (P < 0.05) in females>50y compared with males>50 y, consistent with higher k_uptake values (P < 0.05), with negligible impact on blood exposure (P < 0.05). In males, neither liver exposure, blood exposure, nor k_uptake were affected by aging (P < 0.05). k_uptake was positively and negatively correlated with liver (P < 0.01, R² = 0.78) and blood (P < 0.01, R² = 0.40) exposures respectively. The impact of OATP function (k_uptake) on liver exposure was 4-fold more pronounced than on blood exposure. OATP function may thus drive important sex-related differences in liver exposure, which were not discernible through conventional blood-based pharmacokinetics.