Summary The binding of plasma von Willebrand factor (vWF) to platelet glycoprotein (GP) Ibα in a high shear stress field, and subsequent integrin-GPIIb/IIIa-vWF conjunction induces platelet aggregation (SIPA). However, the specific biomechanical mechanism of the vWF-GPIb interaction still remains to be elucidated. A parallel-plate rectangular flow chamber was built to simulate a stenopeic artery flow pattern. Using the flow chamber, we examined shear- induced platelet activation (SIPAct) at different vWF concentrations (5–25 µg/ml) and several simulated stenotic high shear rates. P-selectin expression on the platelets and annexin V binding to the platelets were used as two markers of platelet activation. At different localized shear rates (3,000 s-1–9,500 s-1), the percentage of annexin V and P-selectin positive cells increased from 8.3 ± 0.4% to 22.3 ± 1.8% ( p 0.05) and from 17.4 ± 0.5% to 33.5 ± 2.5% (p 0.05),respectively. As the vWF concentration increased from 5 µg/ml to 25 µg/ml, the annexinV binding rate increased from 7.2 ± 0.6% to 53.4 ± 3.8% (p 0.05), and P-selectin expression increased from 16.5 ± 1.2% to 65.9 ± 5.2% (p 0.05). A test in a uniform shear field using cone-plate viscometer rheometry showed that the platelet activation rate was proportional to the platelet concentration. This result suggests that platelet collision is one of the impact factors of SIPAct.
The therapeutic utilities of antiangiogenesis and immunotherapy have been proven in clinics, and cancer patients have benefited from respective therapy. Given that the combination of both therapeutic strategies may further improve the effectiveness, a recombinant human 4-1BBL/tumstatin fusion protein (rh4TFP) library was constructed in the present study to target both angiogenesis and T lymphocyte activation, in which the fragments of an endogenous angiogenesis inhibitor tumstatin and a T lymphocyte costimulatory 4-1BBL are coupled with different linkers. After comparison of different combinations, rh4TFP-2 was found to show a promise on potential antiangiogenic immunotherapy. On one hand, rh4TFP-2 inhibited proliferation and migration of human umbilical vein endothelial cells, exhibiting the antiangiogenic activity similar to tumstatin. On the other hand, rh4TFP-2 led to significant increase of T lymphocyte activation for the release of IL-2 and IFN-γ, showing the T lymphocyte activation by 4-1BBL. Moreover, administration of rh4TFP-2 suppressed tumor growth and prolonged survival in a B16F10 melanoma-bearing mouse model. Taken together, the present study provides a new approach of using bifunctional fusion proteins to target both angiogenesis and T lymphocyte activation for cancer therapy.
Abstract Temozolomide (TMZ) therapy offers minimal clinical benefits in patients with glioblastoma multiforme (GBM) with high EGFR activity, underscoring the need for effective combination therapy. Here, we show that tonicity-responsive enhancer binding protein (NFAT5) lysine methylation, is a determinant of TMZ response. Mechanistically, EGFR activation induces phosphorylated EZH2 (Ser21) binding and triggers NFAT5 methylation at K668. Methylation prevents NFAT5 cytoplasm interaction with E3 ligase TRAF6, thus blocks NFAT5 lysosomal degradation and cytosol localization restriction, which was mediated by TRAF6 induced K63-linked ubiquitination, resulting in NFAT5 protein stabilization, nuclear accumulation and activation. Methylated NFAT5 leads to the upregulation of MGMT, a transcriptional target of NFAT5, which is responsible for unfavorable TMZ response. Inhibition of NFAT5 K668 methylation improved TMZ efficacy in orthotopic xenografts and patient-derived xenografts (PDX) models. Notably, NFAT5 K668 methylation levels are elevated in TMZ-refractory specimens and confer poor prognosis. Our findings suggest targeting NFAT5 methylation is a promising therapeutic strategy to improve TMZ response in tumors with EGFR activation.
PD-L2 expression is an important predictor of anti-PD-1 therapy efficacy in patients with head and neck squamous cell carcinoma (HNSCC). However, whether the PD-L2-based immune signature can serve as a prognostic biomarker for patients with HNSCC remains unclear. Here, we reported that PD-L2 was positively stained in 62.7% of tumors, which was more than twice as that of PD-L1, and in 61.4% of patients with PD-L1-negative tumors. Survival tree analysis (STA) revealed that PD-L2high was an independent predictor of poor overall survival (OS). Six patterns were generated from STA, demonstrating that patients with PD-L2lowCD3high were associated with an improved median OS of 72 months and prognostic index (PI) of -3.95 (95% CI, -5.14 to -2.76), whereas patients with PD-L2highCD3lowCD8low to a median OS of 10 months and PI of 1.43 (95% CI, 0.56 to 2.30). Analysis of single-cell RNA sequencing showed that PD-L2 expression was associated with IL-6 expression. We confirmed that IL-6 augments PD-L2 expression in HNSCC cell lines. The PD-L2-based immune signature can serve as an effective biomarker for anti-PD-1 therapy. In addition, PD-L2 may serve as a potential immunotherapeutic target, and we propose anti-IL6 therapy in the adjuvant setting for patients with HNSCC with high PD-L2 expression.
Immune checkpoint blockade therapy targeting programmed cell death protein 1 (PD-1) has revolutionized the landscape of multiple human cancer types, including head and neck squamous carcinoma (HNSCC). Programmed death ligand-2 (PD-L2), a PD-1 ligand, mediates cancer cell immune escape (or tolerance independent of PD-L1) and predicts poor prognosis of patients with HNSCC. Therefore, an in-depth understanding of the regulatory process of PD-L2 expression may stratify patients with HNSCC to benefit from anti-PD-1 immunotherapy. In this review, we summarised the PD-L2 expression and its immune-dependent and independent functions in HNSCC and other solid tumours. We focused on recent findings on the mechanisms that regulate PD-L2 at the genomic, transcriptional, post-transcriptional, translational, and post-translational levels, also in intercellular communication of tumour microenvironment (TME). We also discussed the prospects of using small molecular agents indirectly targeting PD-L2 in cancer therapy. These findings may provide a notable avenue in developing novel and effective PD-L2-targeted therapeutic strategies for immune combination therapy and uncovering biomarkers that improve the clinical efficacy of anti-PD-1 therapies.
Combination therapy has been explored for advanced head and neck squamous cell carcinoma (HNSCC) owing to the limited efficacy of anti-epidermal growth factor receptor (EGFR) therapy. Increased expression and glycosylation of immune checkpoint molecules in tumors are responsible for cetuximab therapy refractoriness. The role of programmed death ligand 2 (PD-L2), a ligand of PD-1, in the immune function is unclear. Here, we examined the regulatory mechanism of PD-L2 glycosylation and its role in antitumor immunity and cetuximab therapy.Single-cell RNA sequencing and immunohistochemical staining were used to investigate PD-L2 expression in cetuximab-resistant/sensitive HNSCC tissues. The mechanism of PD-L2 glycosylation regulation was explored in vitro. The effects of PD-L2 glycosylation on immune evasion and cetuximab efficacy were verified in vitro and using mice bearing orthotopic SCC7 tumors.The PD-L2 levels were elevated and N-glycosylated in patients with cetuximab-resistant HNSCC. Glycosylated PD-L2 formed a complex with EGFR, which resulted in the activation of EGFR/signal transducer and activator of transcription 3 (STAT3) signaling and decreased the cetuximab binding affinity to EGFR. The N-glycosyltransferase fucosyltransferase (FUT8), a transcriptional target of STAT3, was required for PD-L2 glycosylation. Moreover, glycosylation modification stabilized PD-L2 by blocking ubiquitin-dependent lysosomal degradation, which consequently promoted its binding to PD-1 and immune evasion. Inhibition of PD-L2 glycosylation using Stattic, a specific STAT3 inhibitor, or PD-L2 mutation blocking its binding to FUT8, increased cytotoxic T lymphocyte activity and augmented response to cetuximab.Increased expression and glycosylation of PD-L2 in tumors are an important mechanism for cetuximab therapy refractoriness. Thus, the combination of PD-L2 glycosylation inhibition and cetuximab is a potential therapeutic strategy for cancer.
Abstract Temozolomide (TMZ) therapy exert limited clinical benefits in glioblastoma multiforme (GBM) patients with high EGFR activity, underscoring the need for effective combination therapy. Here, we show that tonicity-responsive enhancer binding protein (NFAT5) lysine methylation, is a determinant of TMZ response. Mechanistically, EGFR activation induces phosphorylated EZH2 (Ser21) binds to and triggers NFAT5 methylation at K668. Methylation prevents NFAT5 cytoplasm interaction with E3 ligase TRAF6, thus blocks TRAF6 induced K63-linked ubiquitination mediated NFAT5 lysosomal degradation and cytosol localization restriction, result in NFAT5 protein stabilization, nuclear accumulation and activation. Methylated NFAT5 leads to the upregulation of ITGB1, a transcriptional target of NFAT5, which is responsible for unfavorable TMZ response. Inhibition of NFAT5 K668 methylation improved TMZ efficacy in vivo. Notably, NFAT5 K668 methylation levels are elevated in TMZ-refractory specimens and confer poor prognosis. Our findings suggest targeting NFAT5 methylation as an effective therapeutic strategy to improve TMZ response in tumors with EGFR activation.
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