Changpeng Hu

Background: Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a newly identified immunosuppressive regulator, but its mechanism of suppressing antitumor immunity remains ambiguous. This study aims to uncover the underlying mechanism by which PCSK9 promotes hepatocellular carcinoma (HCC) immune escape and to explore potential intervention strategies. Methods: Co-culture assay assessed the cytotoxicity of CD8 ⁺ T cells against PCSK9-knockout HCC cells. Hepa1-6, H22, and HepG2 cells were used to establish HCC mouse models. Tumor microenvironment changes were evaluated using flow cytometry and single-cell RNA sequencing. Additionally, we developed a CRISPR adenine base editing (ABE) base editor and screened small molecule inhibitors for PCSK9 inhibition in HCC treatment. Results: We found that PCSK9 was highly expressed and correlated with poor survival in patients with HCC. While PCSK9 deficiency did not affect HCC growth in vitro, it significantly enhanced CD8 ⁺ T cell-mediated selective killing in vitro and in vivo. This selective killing of PCSK9-deficient HCC cells could not be explained by existing theories related to major histocompatibility complex-I and T-cell receptor (TCR) degradation. Instead, our study revealed that PCSK9 knockout inhibited the expression of secreted phosphoprotein 1 (SPP1) and programmed death-ligand 1 (PD-L1) in HCC cells, and identified friend leukemia virus integration 1 (FLI1) as their co-transcription factor. Overexpression of FLI1 reversed the PCSK9 knockout-induced downregulation of SPP1 and PD-L1, thereby promoting HCC immune escape. Furthermore, PCSK9 upregulated FLI1 expression through the neurogenic locus notch homolog protein 3 (NOTCH3) pathway. Additionally, we designed an all-in-one ABE base editor with thyroxine-binding globulin promoter (ABE-TBG-PCSK9) to knock down PCSK9 and identified parecoxib as a small molecule inhibitor. We confirmed both approaches enhanced CD8 ⁺ T-cell antitumor activity, significantly inhibiting HCC tumor growth and prolonging mouse survival. Conclusions: PCSK9 promoted HCC immune escape by upregulating SPP1 and PD-L1 via NOTCH3/FLI1 signaling. CRISPR ABE-mediated PCSK9 deficiency and PCSK9 inhibitor parecoxib may serve as effective strategies to inhibit HCC.

Single-molecule mechanochemical sensing (SMMS) is a novel biosensing technique using mechanical force as a signal transduction mechanism. In the mechanochemical sensing, the chemical binding of an analyte molecule to a sensing template is converted to mechanical signals, such as tensile force, of the template. Since mechanical force can be conveniently monitored by single-molecule tools, such as optical tweezers, magnetic tweezers, or Atomic Force Microscopy, mechanochemical sensing is often carried out at the single molecule level. In traditional format of ensemble sensing, sensitivity can be achieved via chemical or electrical amplifications, which are materials intensive and time-consuming. To address these problems, in 2011, we used the principle of mechanochemical coupling in a single molecular template to detect single nucleotide polymorphism (SNP) in DNA fragments. The single-molecule sensitivity in such SMMS strategy allows to removing complex amplification steps, drastically conserving materials and increasing temporal resolution in the sensing. By placing many probing units throughout a single-molecule sensing template, SMMS can have orders of magnitude better efficiency in the materials usage (i.e., high Atom Economy) with respect to the ensemble biosensing. The SMMS sensing probes also enable topochemical arrangement of different sensing units. By placing these units in a spatiotemporally addressable fashion, single-molecule topochemical sensors have been demonstrated in our lab to detect an expandable set of microRNA targets. Because of the stochastic behavior of single-molecule binding, however, it is challenging for the SMMS to accurately report analyte concentrations in a fixed time window. While multivariate analysis has been shown to rectify background noise due to stochastic nature of single-molecule probes, a template containing an array of sensing units has shown ensemble average behaviors to address the same problem. In this so-called ensemble single-molecule sensing, collective mechanical transitions of many sensing units occur in the SMMS sensing probes, which allows accurate quantification of analytes. For the SMMS to function as a viable sensing approach readily adopted by biosensing communities, the future of the SMMS technique relies on the reduction in the complexity and cost of instrumentation to report mechanical signals. In this account, we first explain the mechanism and main features of the SMMS. We then specify basic elements employed in SMMS. Using DNA as an exemplary SMMS template, we further summarize different types of SMMS which present multiplexing capability and increased throughput. Finally, recent efforts to develop simple and affordable high throughput methods for force generation and measurement are discussed in this Account for potential usage in the mechanochemical sensing.

Metastasis is the main cause of death in cancer patients. The efficacy of pharmacological therapy for cancer is limited by the heterogeneous nature of cancer cells and the lack of knowledge of microenvironments in metastasis. Evidence has shown that activated platelets possess both tumor-homing and metastasis-targeting properties via intrinsic cell adhesion molecules on platelets, and malaria protein VAR2CSA is able to specifically bind to oncofetal chondroitin sulfate, which is overexpressed on cancer cells with both epithelial and mesenchymal phenotypes. Inspired by these mechanisms, we developed a recombinant VAR2CSA peptide (rVAR2)-modified activated platelet-mimicking nanoparticles (rVAR2-PM/PLGA-ss-HA) by coating the surface of disulfide-containing biodegradable PLGA conjugate nanoparticles (PLGA-ss-HA) with an activated platelet membrane. The results demonstrated that the engineered 122 nm rVAR2-PM/PLGA-ss-HA inherited the innate properties of the activated platelet membrane and achieved enhanced homing to both primary and metastatic foci. The nanoparticles were endocytosed and responded to a high intracellular concentration of reduced glutathione, resulting in nanoparticle disintegration and the release of chemotherapeutic drugs to kill tumor cells. Thus, rVAR2-decorated activated platelet-targeting nanoparticles with controlled drug release provide a promising drug delivery strategy for efficient treatment of primary and metastatic cancer.

Dopamine deficiency is mainly caused by apoptosis of dopaminergic nerve cells in the substantia nigra of the midbrain and the striatum and is an important pathologic basis of Parkinson’s disease (PD). Recent research has shown that dynamin-related protein 1 (Drp1)-mediated aberrant mitochondrial fission plays a crucial role in dopaminergic nerve cell apoptosis. However, the upstream regulatory mechanism remains unclear. Our study showed that Drp1 knockdown inhibited aberrant mitochondrial fission and apoptosis. Importantly, we found that ROCK1 was activated in an MPP⁺-induced PD cell model and that ROCK1 knockdown and the specific ROCK1 activation inhibitor Y-27632 blocked Drp1-mediated aberrant mitochondrial fission and apoptosis of dopaminergic nerve cells by suppressing Drp1 dephosphorylation/activation. Our in vivo study confirmed that Y-27632 significantly improved symptoms in a PD mouse model by inhibiting Drp1-mediated aberrant mitochondrial fission and apoptosis. Collectively, our findings suggest an important molecular mechanism of PD pathogenesis involving ROCK1-regulated dopaminergic nerve cell apoptosis via the activation of Drp1-induced aberrant mitochondrial fission.

Rho‑associated protein kinase 1 (ROCK1), a member of the ROCK family, serves an important function in cell migration and invasion in neoplasms. ROCK1 has been found to be overexpressed in several types of cancers. However, the role of ROCK1 in non‑small‑cell lung cancer (NSCLC) is poorly understood. In the present study, ROCK1 was found to be overexpressed in NSCLC cells and tissues, and it was associated with poor survival of NSCLC patients. Subsequently, ROCK1 knockdown NSCLC cell lines were established using shRNA. ROCK1 knockdown significantly reduced the migration and invasion ability in the cell monolayer scratching and Transwell assays. ROCK1 knockdown was also found to markedly inhibit cell adhesion ability. Moreover, the phosphorylation of focal adhesion kinase (FAK) was inhibited by ROCK1 knockdown, reducing NSCLC cell migration and invasion ability. This mechanistic study revealed that ROCK1 significantly enhanced cell migration and invasion by inhibiting the phosphatase and tensin homolog (PTEN)/phosphoinositide 3‑kinase (PI3K)/FAK pathway. More importantly, the interruption of the PTEN/PI3K/FAK pathway markedly rescued the inhibition of cell migration and invasion mediated by ROCK1 knockdown. Taken together, these results suggest a novel role for ROCK1 in cell migration and invasion by inhibiting cell adhesion ability, and indicate that ROCK1 may be of value as a therapeutic target for the treatment of NSCLC.

IR‐783 is a kind of heptamethine cyanine dye that exhibits imaging, cancer targeting and anticancer properties. A previous study reported that its imaging and targeting properties were related to mitochondria. However, the molecular mechanism behind the anticancer activity of IR‐783 has not been well demonstrated. In this study, we showed that IR‐783 inhibits cell viability and induces mitochondrial apoptosis in human breast cancer cells. Exposure of MDA‐MB‐231 cells to IR‐783 resulted in the loss of mitochondrial membrane potential (MMP), adenosine triphosphate (ATP) depletion, mitochondrial permeability transition pore (mPTP) opening and cytochrome c (Cyto C) release. Furthermore, we found that IR‐783 induced dynamin‐related protein 1 (Drp1) translocation from the cytosol to the mitochondria, increased the expression of mitochondrial fission proteins mitochondrial fission factor (MFF) and fission‐1 (Fis1), and decreased the expression of mitochondrial fusion proteins mitofusin1 (Mfn1) and optic atrophy 1 (OPA1). Moreover, knockdown of Drp1 markedly blocked IR‐783‐mediated mitochondrial fission, loss of MMP, ATP depletion, mPTP opening and apoptosis. Our in vivo study confirmed that IR‐783 markedly inhibited tumour growth and induced apoptosis in an MDA‐MB‐231 xenograft model in association with the mitochondrial translocation of Drp1. Taken together, these findings suggest that IR‐783 induces apoptosis in human breast cancer cells by increasing Drp1‐mediated mitochondrial fission. Our study uncovered the molecular mechanism of the anti‐breast cancer effects of IR‐783 and provided novel perspectives for the application of IR‐783 in the treatment of breast cancer.