BACKGROUND: Cervical squamous cell carcinoma (CESC), the most common subtype of cervical cancer, is primarily caused by the high-risk human papillomavirus (HPV) infection and genetic susceptibility. Single-cell RNA sequencing (scRNA-seq) has been widely used in CESC research to uncover the diversity of cell types and states within tumor tissues, enabling a detailed study of the tumor microenvironment (TME). This technology allows precise mapping of HPV infection in cervical tissues, providing valuable insights into the initiation and progression of HPV-mediated malignant transformation. METHODS: We performed the scRNA-seq to characterize gene expression in tumor tissues and paired adjacent para-cancerous tissues from four patients with early-stage CESC using the 10× Genomics platform. The HPV infection and its subtypes were identified using the scRNA data and viral sequence mapping, and trajectory analyses were performed using HPV+ or HPV- cells. Interactions between different types of keratinized cells and their interactions with other cell types were identified, and pathways and specificity markers were screened for proliferating keratinized cells. The Cancer Genome Atlas (TCGA) dataset was used to verify the prognostic correlation between tumor-specific PI3+S100A7+ keratinocyte infiltration and CESC, and the localization relationship between PI3+S100A7+ keratinocytes and macrophages was verified by immunofluorescence staining. RESULTS: Various types of keratinocytes and fibroblasts were the two cell types with the most significant differences in percentage between the tumor tissue samples and paired adjacent non-cancerous tissue samples in the early stages of CESC. We found that PI3+S100A7+ keratinocytes were associated with early HPV-positive CESC, and PI3+S100A7+ keratinocytes were more abundant in tumors than in adjacent normal tissues in the TCGA-CESC dataset. Analysis of clinical information revealed that the infiltration of PI3+S100A7+ keratinocytes was notably higher in tumors with poor prognosis than in those with good prognosis. Additionally, multiplex immunofluorescence analysis showed a specific increase in PI3+S100A7+ expression within tumor tissues, with PI3+S100A7+ keratinocytes and CD163+ macrophages being spatially very close to each other. In the analysis of cell-cell interactions, macrophages exhibited strong crosstalk with PI3+S100A7+ proliferating keratinocytes in HPV-positive CESC tumors, mediated by tumor necrosis factor (TNF), CCL2, CXCL8, and IL10, highlighting the dynamic and tumor-specific enhancement of macrophage-keratinocyte interactions, which are associated with poor prognosis and immune modulation. Using CIBERSORTx, we discovered that patients with high infiltration of both PI3+S100A7+ proliferating keratinocytes and macrophages had the shortest overall survival. In the analysis of cell-cell interactions, PI3+S100A7+ proliferating keratinocytes and macrophages were found to be involved in highly active pathways that promote differentiation and structure formation, including cytokine receptor interactions, the Nuclear Factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling pathway, and TNF signaling pathway regulation. Further subtyping of fibroblast populations identified four subtypes. The C1 group, characterized by its predominance in tumor tissues, is a subtype enriched with cancer-associated fibroblasts (CAFs), whereas the C3 group is primarily enriched in adjacent non-cancerous tissues and consists of undifferentiated cells. Moreover, the distinct molecular and cellular differences between HPV16- and HPV66-associated tumors were demonstrated, emphasizing the unique tumor-promoting mechanisms and microenvironmental influences driven by each HPV subtype. CONCLUSIONS We discovered a heterogeneous population of keratinocytes between tumor and adjacent non-cancerous tissues caused by HPV infection and identified macrophages and specific CAFs that play a crucial role during the early stage in promoting the inflammatory response and remodeling the cancer-promoting TME. Our findings provide new insights into the transcriptional landscape of early-stage CESC to understand the mechanism of HPV-mediated malignant transformation in cervical cancer.
Humans ; Female ; Papillomavirus Infections/genetics* ; Uterine Cervical Neoplasms/genetics* ; Carcinoma, Squamous Cell/pathology* ; Keratinocytes/metabolism* ; Single-Cell Analysis/methods* ; Tumor Microenvironment/genetics*

The NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome, a high-molecular-weight protein complex in the cytoplasm, is composed of three core components: the sensor protein NLRP3, the adaptor protein apoptosis-associated speck-like protein containing a caspase-recruitment domain (ASC) and the effector protein caspase-1. It plays a critical role in regulating host immune and inflammatory responses. Studies have shown that the NLRP3 inflammasome has increasingly become a focal point in tumor molecular biology field. A growing body of evidence indicates that the increased expression and activation of the NLRP3 inflammasome is closely associated with the pathogenesis of head and neck squamous cell carcinoma (HNSCC) and the tumor microenvironment (TME). It may promote tumor proliferation, invasion, migration, and other biological behaviors through various regulatory mechanisms while influencing tumor immune evasion and therapy resistance, which holds promise as a prognostic biomarker for patients. This review explores the current effect and mechanism of the NLRP3 inflammasome and its signaling pathways in head and neck cancer, providing insights into clinical targeted drug development and molecular immunotherapy.
Humans ; NLR Family, Pyrin Domain-Containing 3 Protein/genetics* ; Inflammasomes/metabolism* ; Head and Neck Neoplasms/pathology* ; Squamous Cell Carcinoma of Head and Neck/metabolism* ; Tumor Microenvironment ; Signal Transduction ; Animals

OBJECTIVES: Esophageal squamous cell carcinoma (ESCC) is characterized by complex pathogenesis and poor prognosis. In recent years, epithelial-mesenchymal transition (EMT) in tumor initiation and progression has attracted increasing attention. Enhancer of zeste homolog 2 (EZH2), which is aberrantly expressed in various tumors, may be closely related to the EMT process. This study aims to examine the expression and correlation of EZH2 and EMT markers in ESCC cells and tissues, evaluate the effects of EZH2 knockdown on ESCC cell proliferation, invasion, and migration, and explore how EZH2 contributes to the malignant biological behavior of ESCC. METHODS: Bioinformatics analyses were used to assess EZH2 expression levels in ESCC. Small interfering RNA was used to knock down EZH2 in ESCC cell lines EC109 and EC9706. Cell proliferation, invasion, and migration were evaluated using cell counting kit-8 (CCK-8), wound healing, and Transwell assays. Protein and mRNA expression levels of EZH2, E-cadherin (E-cad), and vimentin (Vim) were detected by Western blotting and real time fluorogenic quantitative PCR (RT-qPCR), respectively. Immunohistochemical (IHC) staining was performed on 70 ESCC tissue samples and 40 paired adjacent normal tissues collected from the First Affiliated Hospital of Shihezi University between 2010 and 2016 to assess the expression of EZH2, E-cad, and Vim, and to analyze their associations with clinicopathological feature and patient prognosis. RESULTS: Bioinformatics analysis showed that EZH2 was highly expressed in ESCC (P<0.001), and high EZH2 expression was associated with worse prognosis (P<0.001). CCK-8, wound healing, and Transwell assays demonstrated that EZH2 knockdown significantly suppressed the proliferation, invasion, and migration of ESCC cells (P<0.001). In addition, Vim expression was significantly reduced, while E-cad expression was significantly increased at both protein and mRNA levels in EZH2-silenced cells (all P<0.05). IHC staining analysis revealed higher expression of EZH2 and Vim and lower expression of E-cad in ESCC tissues compared to adjacent normal tissues. Kaplan-Meier survival analysis showed that low expression of EZH2 and Vim and high expression of E-cad were associated with longer survival (all P<0.05). CONCLUSIONS EZH2 promotes malignant biological behavior in ESCC by mediating EMT. Elevated EZH2 expression is associated with poor prognosis in ESCC patients.
Humans ; Enhancer of Zeste Homolog 2 Protein/physiology* ; Esophageal Squamous Cell Carcinoma/pathology* ; Epithelial-Mesenchymal Transition/genetics* ; Esophageal Neoplasms/metabolism* ; Cell Proliferation ; Cell Line, Tumor ; Cell Movement ; Cadherins/genetics* ; Vimentin/genetics* ; Male ; Female ; Middle Aged ; Neoplasm Invasiveness ; Prognosis ; RNA, Small Interfering/genetics* ; Gene Expression Regulation, Neoplastic

Mitochondria are the convergence point of multiple pathways that trigger programmed cell death (PCD). Mitochondrial-associated PCD (mtPCD) is involved in the pathogenesis of several diseases. However, the role of mtPCD in the prognostic prediction of cancers including non-small-cell lung cancer (NSCLC) remains to be investigated. Here, 12 mtPCD patterns were analyzed in transcriptomics, genomics, and clinical data collected from 4 datasets containing 977 patients. A risk-score assessment system containing 18 genes was established. We found that NSCLC patients with a high-risk score had a poorer prognosis. A nomogram was constructed by incorporating the risk score with clinical features. The risk score was further associated with clinicopathological information, tumor-mutation frequency, and immunotherapy responses. NSCLC patients with a high risk score had more Treg cells infiltration. However, these patients had higher tumor-mutation burden scores and may be more sensitive to immunotherapy. Moreover, receptor-interacting serine/threonine protein kinase 2 (RIPK2) was selected from mtPCD gene model for validation. We found that RIPK2 exhibited oncogenic function, and its expression level was inversely associated with the overall survival of NSCLC. Taken together, our results indicated the accuracy and practicability of the mtPCD gene model and RIPK2 in predicting the prognosis of NSCLC.
Humans ; Carcinoma, Non-Small-Cell Lung/pathology* ; Lung Neoplasms/pathology* ; Prognosis ; Male ; Female ; Nomograms ; Middle Aged ; Mitochondria/metabolism* ; Apoptosis/genetics* ; Mutation ; Biomarkers, Tumor/genetics* ; Aged

OBJECTIVES: To investigate the role of Holliday cross-recognition protein (HJURP) in tumorigenesis, progression, and immunotherapy responses. METHODS: Bioinformatics approaches were used to analyze the expression level of HJURP in various cancers and its association with prognosis, clinical stage, and immune cell infiltration using TCGA, GTEx, SangerBox and TIMER 2.0 databases. LinkedOmics database was employed to investigate HJURP-related genes and their potential functions in kidney renal clear cell carcinoma (KIRC). The expression of HJURP in KIRC samples was examined with immunohistochemistry, Western blotting and qRT-PCR, and the effect of HJURP silencing on cell proliferation and migration was tested in cultured KIRC cells. RESULTS: HJURP was highly expressed in 26 cancers with negative correlations with the patients' survival outcomes in 5 cancers including KIRC (P<0.05). HJURP expression levels was strongly correlated with clinical stages and immune cell infiltration in the tumors. In KIRC, HJURP expression was significantly elevated (P<0.0001) and showed a positive correlation with TNM stage (P<0.05), overall stage (P<0.01) and immune cell infiltration. Gene Ontology (GO) functional analysis showed that HJURP is predominantly enriched in biological processes such as biological regulation and metabolic processes. Concerning cellular components, HJURP is primarily localized to the cell membrane and nucleus. In terms of molecular functions, it is chiefly enriched in activities related to protein binding and ion binding. HJURP was highly expressed in both clinical KIRC tissues and KIRC cell lines (P<0.001). In cultured KIRC cells, silencing of HJURP significantly inhibited cell proliferation and migration abilities. CONCLUSIONS HJURP may serves as an indicator of prognosis and immunotherapy response of KIRC, and its high expression enhances malignant behaviors of KIRC cells.
Humans ; Prognosis ; Kidney Neoplasms/pathology* ; Biomarkers, Tumor/metabolism* ; Carcinoma, Renal Cell/pathology* ; DNA-Binding Proteins/genetics* ; Cell Proliferation ; Cell Line, Tumor ; Cell Movement

Dysregulated RNA splicing events produce transcripts that facilitate esophageal squamous cell carcinoma (ESCC) progression, but how this splicing process is abnormally regulated remains elusive. Here, we unveiled a novel alternative splicing axis of BOLA3 transcripts and its regulator HNRNPC in ESCC. The long-form BOLA3 (BOLA3-L) containing exon 3 exhibited high expression levels in ESCC and was associated with poor prognosis. Functional assays demonstrated the protumorigenic function of BOLA3-L in ESCC cells. Additionally, HNRNPC bound to BOLA3 mRNA and promoted BOLA3 exon 3 inclusion forming BOLA3-L. High HNRNPC expression was positively correlated with the presence of BOLA3-L and associated with an unfavorable prognosis. HNRNPC knockdown effectively suppressed the malignant biological behavior of ESCC cells, which were significantly rescued by BOLA3-L overexpression. Moreover, BOLA3-L played a significant role in mitochondrial structural and functional stability. E2F7 acted as a key transcription factor that promoted the upregulation of HNRNPC and inclusion of BOLA3 exon 3. Our findings provided novel insights into how alternative splicing contributes to ESCC progression.
Female ; Humans ; Male ; Mice ; Alternative Splicing ; Cell Line, Tumor ; Disease Progression ; Esophageal Neoplasms/pathology* ; Esophageal Squamous Cell Carcinoma/pathology* ; Exons/genetics* ; Gene Expression Regulation, Neoplastic ; Heterogeneous-Nuclear Ribonucleoprotein Group C/metabolism* ; Prognosis ; RNA, Long Noncoding/metabolism* ; Animals

Objective: To investigate the clinicopathological features, treatment and prognosis of patients with RET fusion positive non-small cell lung cancer (NSCLC). Methods: A total of 1 089 NSCLCs were retrieved at Affiliated Hospital of Jiangnan University from August 2018 to April 2020. In all cases, multiple gene fusion detection kits (fluorescent PCR method) were used to detect the gene status of RET, EGFR, ALK, ROS1, KRAS, BRAF and HER2; and immunohistochemical method was used to detect the expression of PD-L1 and mismatch repair related proteins. The correlation between RET-fusion and patients' age, gender, smoking history, tumor stage, grade, pathologic type, and PD-L1, mismatch repair related protein expression was analyzed. Results: There were 22 cases (2.02%) detected with RET fusion-positive in 1 089 NSCLC patients, in which 11 males and 11 females; and the median age was 63.5 years. There were 20 adenocarcinomas, including 11 acinar predominant adenocarcinoma (APA), five solid predominant adenocarcinoma (SPA) and four lepidic predominant adenocarcinoma (LPA); There were one case each of squamous cell carcinoma (non-keratinizing type) and sarcomatoid carcinoma (pleomorphic carcinoma). There were 6 and 16 patients with RET fusion-positive who were in stage Ⅰ-Ⅱ and Ⅲ-Ⅳ respectively, and 16 cases with lymph node metastasis, 11 cases with distant metastasis. Among RET fusion-positive cases, one was detected with HER2 co-mutation. The tumor proportion score of PD-L1≥1% in patients with RET fusion positive lung cancer was 54.5% (12/22). Defects in mismatch repair protein expression were not found in patients with RET fusion positive NSCLC. Four patients with RET fusions positive (two cases of APA and two cases of SPA) received pratinib-targeted therapy, and two showed benefits from this targeted therapy. Conclusions: The histological subtypes of RET fusions positive NSCLC are more likely to be APA or SPA. RET fusion-positive NSCLC patients are associated with advanced clinical stage, lymph node metastases, and they may benefit from targeted therapy with RET-specific inhibitors.
Male ; Female ; Humans ; Middle Aged ; Carcinoma, Non-Small-Cell Lung/pathology* ; Lung Neoplasms/pathology* ; B7-H1 Antigen/genetics* ; Protein-Tyrosine Kinases/genetics* ; Proto-Oncogene Proteins c-ret/metabolism* ; Proto-Oncogene Proteins/genetics* ; Adenocarcinoma/pathology* ; Carcinoma, Squamous Cell/genetics* ; Mutation

OBJECTIVE: Programmed cell death 6 (PDCD6), a Ca ²⁺-binding protein, has been reported to be aberrantly expressed in all kinds of tumors. The aim of this study was to explore the role and mechanism of PDCD6 in hepatocellular carcinomas (HCCs). METHODS: The expression levels of PDCD6 in liver cancer patients and HCC cell lines were analyzed using bioinformatics and Western blotting. Cell viability and metastasis were determined by methylthiazol tetrazolium (MTT) and transwell assays, respectively. And Western blotting was used to test related biomarkers and molecular pathway factors in HCC cell lines. LY294002, a PI3K inhibitor inhibiting AKT, was used to suppress the AKT/GSK3β/β-catenin pathway to help evaluate the role of this pathway in the HCC carcinogenesis associated with PDCD6. RESULTS: The analysis of The Cancer Genome Atlas Database suggested that high PDCD6 expression levels were relevant to liver cancer progression. This was consistent with our finding of higher levels of PDCD6 expression in HCC cell lines than in normal hepatocyte cell lines. The results of MTT, transwell migration, and Western blotting assays revealed that overexpression of PDCD6 positively regulated HCC cell proliferation, migration, and invasion. Conversely, the upregulation of PDCD6 expression in the presence of an AKT inhibitor inhibited HCC cell proliferation, migration, and invasion. In addition, PDCD6 promoted HCC cell migration and invasion by epithelial-mesenchymal transition. The mechanistic investigation proved that PDCD6 acted as a tumor promoter in HCC through the AKT/GSK3β/β-catenin pathway, increasing the expression of transcription factors and cellular proliferation and metastasis. CONCLUSION PDCD6 has a tumor stimulative role in HCC mediated by AKT/GSK3β/β-catenin signaling and might be a potential target for HCC progression.
Humans ; Carcinoma, Hepatocellular/pathology* ; Liver Neoplasms/pathology* ; Proto-Oncogene Proteins c-akt/metabolism* ; beta Catenin/metabolism* ; Phosphatidylinositol 3-Kinases/metabolism* ; Glycogen Synthase Kinase 3 beta/metabolism* ; Cell Line ; Cell Proliferation ; Cell Line, Tumor ; Gene Expression Regulation, Neoplastic ; Calcium-Binding Proteins/metabolism* ; Apoptosis Regulatory Proteins/genetics*

BACKGROUND: There have been many significant advances in the diagnosis and treatment of non-small cell lung cancer (NSCLC). However, the mechanism underlying the progression of NSCLC is still not clear. Plant homodomain finger-like domain-containing protein 5A (PHF5A) plays an important role in processes of chromatin remodeling, morphological development of tissues and organs and maintenance of stem cell pluripotency. This study aims to investigate the role of PHF5A in the proliferation and migration of NSCLC. METHODS: A549 and PC-9 PHF5A overexpression cell lines were constructed. PHF5A expression was decreased in H292 and H1299 cells by using siRNA. Flow cytometry was used to detect the cell cycle. MTT assay and clone formation assay were used to examine the proliferative ability of NSCLC, while migration assay and wound healing assay were performed to evaluate the ability of migration. Western blot analysis was used to measure the expressions of PI3K, p-AKT and the associated downstream factors. RESULTS: Up-regulation of PHF5A in A549 and PC-9 cells increased the proliferation rate, while down-regulation of PHF5A in H292 and H1299 cells inhibited the proliferation rate at 24 h, 48 h and 72 h (P<0.05). The metastatic ability was elevated in the PHF5A-overexpresion groups, while reduced in the PHF5A-down-regulation group (P<0.05). In addition, reduced expression of PHF5A induced cell cycle arrest at G1/S phase (P<0.05). Furthermore, decreased expression of PHF5A reduced the expression levels of PI3K, phosphorylation of AKT, c-Myc (P<0.05) and elevated the expression of p21 (P<0.05). CONCLUSIONS These results demonstrated that PHF5A may play an important role in progression of NSCLC by regulating the PI3K/AKT signaling pathway.
Humans ; Carcinoma, Non-Small-Cell Lung/pathology* ; Lung Neoplasms/pathology* ; Proto-Oncogene Proteins c-akt/metabolism* ; Phosphatidylinositol 3-Kinases/metabolism* ; Cell Line, Tumor ; Cell Proliferation/genetics* ; Cell Movement/genetics* ; Gene Expression Regulation, Neoplastic ; Trans-Activators/genetics* ; RNA-Binding Proteins/metabolism*

Lung squamous cell carcinoma (LSCC) accounts for approximately 30% of non-small cell lung cancer (NSCLC) cases and is the second most common histological type of lung cancer. Anaplastic lymphoma kinase (ALK)-positive NSCLC accounts for only 2%-5% of all NSCLC cases, and is almost exclusively detected in patients with lung adenocarcinoma. Thus, ALK testing is not routinely performed in the LSCC population, and the efficacy of such treatment for ALK-rearranged LSCC remains unknown. Echinoderm microtubule associated protein like 4 (EML4)-ALK (V1) and TP53 co-mutations were identified by next generation sequencing (NGS) in this patient with advanced LSCC. On December 3, 2020, Ensatinib was taken orally and the efficacy was evaluated as partial response (PR). The progression-free survival (PFS) was 19 months. When the disease progressed, the medication was changed to Loratinib. To our knowledge, Enshatinib created the longest PFS of ALK-mutant LSCC patients treated with targeted therapy since literature review. Herein, we described one case treated by Enshatinib involving a patient with both EML4-ALK and TP53 positive LSCC, and the relevant literatures were reviewed for discussing the treatment of this rare disease.
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Humans ; Carcinoma, Non-Small-Cell Lung/drug therapy* ; Lung Neoplasms/pathology* ; Anaplastic Lymphoma Kinase/metabolism* ; Carcinoma, Squamous Cell/genetics* ; Mutation ; Cytoskeletal Proteins/genetics* ; Lung/pathology* ; Oncogene Proteins, Fusion/genetics* ; Protein Kinase Inhibitors/therapeutic use* ; Tumor Suppressor Protein p53/genetics*