Inhibition of YAP Sensitizes the Selumetinib Treatment for Neurofibromatosis Type 1 Related Plexiform Neurofibroma.

Background: Targeted therapy of Neurofibromatosis type 1 (NF1) related plexiform neurofibroma (pNF) aiming at MEK molecule has not demonstrated a convincing result for complete disease inhibition, probably due to other signal pathways crosstalk. Our previous study revealed an increased nuclear translocation of YAP molecule in NF1 related pNF. Herein, we decided to further investigate the therapeutic relations of YAP interference during the MEK treatment against NF1 related pNF. Methods: By means of selumetinib (MEK-inhibitor), RNA-sequencing was firstly performed to identify the changes of signal pathways in pNF Schwann cells, which was probably related to YAP regulation. Nuclear-cytoplasmic fractionation and western blotting were performed to show the intracellular YAP changes under selumetinib treatment. Thirdly, a series of in vitro assays were performed including flow cytometry, CCK-8, and colony/sphere formation under dual treatment of selumetinib and verteporfin (YAP-inhibitor). In addition, Chou-Talalay method was adopted to evaluate the synergistic inhibiting effects of such drug combination. Xenograft study was also used to detect the combining effects in vivo. Results: RNA-sequencing revealed that selumetinib treatment might be associated with the undesirable activation of Hippo pathway in NF1 related pNF tumor cells, which might reduce its pharmaceutic effects. Next, nuclear-cytoplasmic fractionation and further studies demonstrated that selumetinib could promote the nuclear translocation and transcriptional activation of YAP in vitro, which might cause the aforementioned resistance to selumetinib treatment. Additionally, when combined treatments were performed based on verteporfin and selumetinib, synergistic effects were observed on cytotoxicity of NF1 related pNF tumor cells in vitro and in vivo xenograft models. Conclusion: YAP inhibition can effectively sensitize NF1 related pNF tumor cells to selumetinib. Dual targeting of YAP and MEK might be a promising therapeutic strategy for treating NF1 related pNF.

Schwann cell derived pleiotrophin stimulates fibroblast for proliferation and excessive collagen deposition in plexiform neurofibroma.

Neurofibromatosis type 1 associated plexiform neurofibroma (pNF) is characterized by abundant fibroblasts and dense collagen, yet the intricate interactions between tumor-origin cells (Schwann cells) and neurofibroma-associated fibroblasts (NFAFs) remain elusive. Employing single-cell RNA sequencing on human pNF samples, we generated a comprehensive transcriptomics dataset and conducted cell-cell communication analysis to unravel the molecular dynamics between Schwann cells and NFAFs. Our focus centered on the pleiotrophin (PTN)/nucleolin (NCL) axis as a pivotal ligand-receptor pair orchestrating this interaction. Validation of PTN involvement was affirmed through coculture models and recombinant protein experiments. Functional and mechanistic investigations, employing assays such as CCK8, EdU, Western Blot, ELISA, Hydroxyproline Assay, and Human phospho-kinase array, provided critical insights. We employed siRNA or inhibitors to intercept the PTN/NCL/proline-rich Akt substrate of 40 kDa (PRAS40) axis, validating the associated molecular mechanism. Our analysis highlighted a subset of Schwann cells closely linked to collagen deposition, underscoring their significance in pNF development. The PTN/NCL axis emerged as a key mediator of the Schwann cell-NFAF interaction. Furthermore, our study demonstrated that elevated PTN levels enhanced NFAF proliferation and collagen synthesis, either independently or synergistically with TGF-ß1 in vitro. Activation of the downstream molecule PRAS40 was noted in NFAFs upon PTN treatment. Crucially, by targeting NCL and PRAS40, we successfully reversed collagen synthesis within NFAFs. In conclusion, our findings unveil the pivotal role of the PTN/NCL/PRAS40 axis in driving pNF development by promoting NFAFs proliferation and function. Targeting this pathway emerges as a potential therapeutic strategy for pNF. This study contributes novel insights into the molecular mechanisms governing pNF pathogenesis.

Identification of Exosome-Related Genes Associated with Prognosis and Immune Infiltration Features in Head-Neck Squamous Cell Carcinoma.

The highly immunosuppressive nature of head-neck squamous cell cancer (HNSCC) is not fully understood. Exosomes play crucial roles in the communication between cancer and non-cancer cells, but the clinical significance of the expression of exosome-related genes (ERGs) remains unclear in HNSCC. This study aimed to establish an HNSCC-ERGs model by using mass spectrometry (MS)-based label-free quantitative proteomics in combination with the TCGA primary HNSCC dataset. The study managed to classify the HNSCC patients into two subtypes based on the expression level of prognostic ERGs, which showed significant differences in prognosis and immune infiltration. LASSO regression algorithm was used to establish a risk prediction model based on seven risky genes (PYGL, ACTN2, TSPAN15, EXT2, PLAU, ITGA5), and the high-risk group was associated with poor survival prognosis and suppressive immune status. HPRT1 and PYGL were found to be independent prognostic factors through univariate and multivariate Cox regression analyses. Immune and ssGSEA analysis revealed that HPRT1 and PYGL were significantly related to immunosuppression, immune response, and critical signaling transduction pathways in HNSCC. Immunohistochemistry results further validated the expression level, clinical value, and immunosuppressive function of HPRT1 and PYGL in HNSCC patients. In conclusion, this study established molecular subtypes and a prediction risk model based on the ERGs. Furthermore, the findings suggested that HPRT1 and PYGL might play critical roles in reshaping the tumor microenvironment.

M1-like tumor-associated macrophages cascade a mesenchymal/stem-like phenotype of oral squamous cell carcinoma via the IL6/Stat3/THBS1 feedback loop.

BACKGROUND: Tumor-associated macrophages (TAMs) have a leading position in the tumor microenvironment. Previously, we have demonstrated that M1-like TAMs activated by exosome-transferred THBS1 promote malignant migration in oral squamous cell carcinoma (OSCC). However, the functional roles and associated molecular mechanisms of the activated M1-like TAMs need to be further clarified in OSCC. METHODS: Conditioned Media (CM) were harvested from the exosome activated M1-like TAMs. We measured the malignant behaviors of OSCC under the treatment of CM from M1-like TAMs by performing colony forming assays, invasion assays, wound-healing assays, spheroid forming assays and in vivo xenograft experiments. The underlying mechanisms were investigated by RNA-seq, cytokines analysis, intracellular signaling pathway analysis, ChIP assays, bioinformatics analysis and validation. RESULTS: M1-like TAMs significantly promoted the epithelial-mesenchymal transition (EMT) process, and induced the cancer-stem like cells (CSCs) by upregulating the expression of MME and MMP14 in OSCC cells. Cytokine analysis revealed a shark increase of IL6 secretion from M1-like TAMs. Blocking IL6 in the CM from M1-like TAMs could significantly weaken its effects on the colony forming, invasion, migration, microsphere forming and xenograft forming abilities of OSCC cells. Cellular signaling assays indicated the activation of Jak/Stat3 pathway in the OSCC cells treated by the CM from M1-like TAMs. Blocking the activation of the Jak/Stat3 pathway could significantly weaken the effects of M1-like TAMs on the colony forming, invasion, migration, microsphere forming and xenograft forming abilities of OSCC cells. Further RNA-seq analysis and bioinformatics analysis revealed an increased expression of THBS1 in the OSCC cells treated by M1-like TAMs. Bioinformatics prediction and ChIP assays revealed the activation of Stat3 by CM from M1-like TAMs could directly promote the transcription of THBS1 in OSCC cells. CONCLUSIONS: We proposed that M1-like TAMs could cascade a mesenchymal/stem-like phenotype of OSCC via the IL6/Stat3/THBS1 feedback loop. A better understanding on the functional roles and associated molecular mechanisms of M1-like TAMs might facilitate the development of novel therapies for supplementing the current treatment strategies for OSCC patients.

Bioinformatics analysis for the biomarkers of the tumor-infiltrating lymphocytes in head and neck squamous cell carcinoma.

BACKGROUND: Immunotherapy strategies are successful in only a subset of patients with advanced head and neck squamous cell carcinoma (HNSCC). The roles of tumor-infiltrating lymphocytes (TILs) in HNSCC are less clear. Herein, we present a preliminary study to identify the underlying heterogeneity and correlations among TILs in HNSCC by bioinformatics analysis of TIL-related biomarkers. METHODS: The expression patterns, clinicopathological values and underlying correlations for the TILs related genes were analyzed based on the TCGA primary HNSCC cohort. The prognostic significance for the involved genes in the HNSCC patients was evaluated by using an online tool, Kaplan-Meier Plotter. Bioinformatics prediction for the co-expression, physical interactions, pathway, and genetic interactions of the involved genes was performed by using GeneMANIA. RESULTS: The expression of programmed death-ligand 1 (PD-L1) was significantly correlated with the expression of CD4 (P<0.01), CD8 (P<0.01), and CD20 (P=0.011), but not CD56 (P=0.065) based on the TCGA primary HNSCC cohort. For the expression of programmed cell death 1 (PD-1), a significant correlation was also observed with the expression of CD4 (P<0.01), CD8 (P<0.01), and CD20 (P<0.01), but not CD56 (P=0.861). For the clinically significant evaluation, variable roles for the biomarkers were compared and demonstrated. Increased expression of CD8 (P=0.029), CD20 (P<0.01), or PD-1 (P=0.0084) significantly correlated with improved overall survival (OS), and increased CD56 expression indicated obviously decreased OS for HNSCC patients (P=0.0033). Significantly positive correlations between human papilloma virus (HPV) status and the expression of CD8 (P<0.01), CD20 (P<0.01) and PD-1 (P<0.01) were demonstrated and a significantly negative correlation between HPV status and CD56 expression was also demonstrated (P<0.01). In addition, IL2RB was determined to be a hub factor that regulates the infiltration of TILs and the expression of PD-1/PD-L1 in HNSCC. CONCLUSIONS: A systematic evaluation for the TILs status can be informative to predict prognosis and direct the immunotherapy for HNSCC patients, but further investigations are still needed.

Primary undifferentiated pleomorphic sarcoma in oral-maxillary area: retrospective study and molecular analysis.

Undifferentiated pleomorphic sarcoma (UPS) in oral-maxillary area is rarely reported. Herein, we aimed to investigate the clinical characteristics, treatment strategies, prognosis, and molecular features of the oral-maxillary UPS. In total, 10 cases with primary oral-maxillary UPS were included. The rapidly progressive UPS can easily develop to an advanced and life-threatening stage, especially concerning the complex anatomical structures and spaces in the oral-maxillary area. The final diagnosis for UPS greatly depended on histological findings and immunohistochemistry staining after the exclusion of all possible differential diagnoses. Retrospectively, the treatment strategies for the included cases still referred to those of oral squamous cell carcinoma (OSCC). Statistically, the median overall survival (OS) for all the included cases was 7.75 months (range: 5-17 months). Comparatively, 3 cases had improved OS (median survival: 17 months, range: 17-18 months) and experienced PR/SD with neoadjuvant chemotherapy (anlotinib). The molecular features were demonstrated by using whole exonic sequencing for 1 included case. Cancer driver gene detection revealed GBP4 as a candidate driver gene for the primary oral-maxillary UPS. Additionally, a missense mutation in gene PIK3CA (p.E545K) was also identified. Our findings could greatly expand the knowledge about primary oral-maxillary UPS, and provide molecular evidences to improve the therapeutic options for primary oral-maxillary UPS.