Acta Histochemica

Immunohistochemistry and machine learning study of DNA replication-associated proteins in uterine epithelial tumors and precursor lesions

Endometrioid adenocarcinoma (EA) has been on the increase in recent years in developed countries. Early detection of endometrioid adenocarcinoma in the endometrial corpus is crucial for patient prognosis and early treatment, although their distinction can sometimes be challenging. In this study, we focused on DNA replication-related proteins through immunohistochemical analysis and investigated whether the discrimination between EA and their precursor lesions is achievable using machine learning techniques. The research utilized tissue specimens from 100 cases, including EA of different grades (Grade 1; G1, Grade 2; G2, Grade 3; G3) and their precursor lesions (endometrial hyperplasia without atypia; EH, endometrial atypical hyperplasia: AH). Immunohistochemical analysis of DNA replication-related proteins, such as ORC1, Cdt1, Cdc6, MCM7, Cdc7, and Geminin, was conducted for each case, measuring the Labeling Index (LI) and optical density (OD) of protein expression. Furthermore, we performed statistical significance tests and machine learning -discriminant analysis using LI and OD as inputs, employing non-linear Support Vector Machines (NSVM). The NSVM discriminant analysis demonstrated the accuracy of over 85 % between EH and each differentiation grade of EA, the accuracy is also similar for AH and each differentiation grade of EA. In addition, changing the combination of DNA replication-related proteins used for discrimination resulted in a high accuracy (95-100 %). A discriminant analysis with NSVM using the LI and OD of DNA replication-related proteins may enable the differentiation of EA from its precursor lesions.

Nuclear expression of NHERF1/EBP50 in Clear Cell Renal Cell Carcinoma

The Na/H exchange regulatory factor 1 or Ezrin-radixin-moesin-binding phosphoprotein 50 (NHERF1/EBP50) is an adaptor protein implicated in the stabilization of molecular complexes linking extracellular signals with the cytoskeleton machinery. NHERF1 expression at the cell cortex is associated with the maintenance of adherent junction integrity in polarized epithelia. The role of NHERF1 in cancer depends on its localization within the cell, acting, in most cases, as a tumor suppressor when localized at the cell membrane, and as an oncogene, when expressed in the cytoplasm or the nucleus of cancer cells. The distribution of NHERF1 in renal cell carcinoma (RCC) has not been yet investigated. In this study, NHERF1 expression was examined by immunohistochemistry in papillary and clear cell RCC. We observed membranous staining in papillary RCC, whereas NHERF1 expression was nuclear and membranous in clear cell RCC. In comparison, NHERF1 immunohistochemistry in clear cell carcinomas of the ovary showed mainly nuclear staining. Our finding of the specific NHERF1 nuclear expression in clear cell carcinomas may help to elucidate the molecular changes that regulate its nuclear accumulation and to better understand its role in this cell compartment.

Inhibition of Lonp1 induces mitochondrial remodeling and autophagy suppression in cervical cancer cells

Lon protease 1(Lonp1) is an ATP-dependent protease located in the mitochondrial matrix and plays a crucial role in preserving normal mitochondrial function. Lonp1 overexpression is associated with tumorigenesis in various cancer types, including cervical cancer. In the present study, we show that the Lonp1 content is elevated in cervical cancer tissues compared to cervical paracancerous tissues. Conversely, Lonp1 knockdown suppresses cervical cancer cell proliferation, migration and invasion but promotes apoptosis. Mechanistically, Lonp1 knockdown decreases area of mitochondrial networks and induces mitochondrial depolarization. Furthermore, Lonp1 inhibition reduces the level of LC3-II/I, PINK1 and Parkin, but promotes the level of p62. Collectively, our study suggests that the anti-cancer effect caused by Lonp1 downregulation likely contributes to mitochondrial remodeling and suppression of autophagy and mitophagy.

Quantifying mechanical, proliferation, and migrational properties of bulk ovarian cancer cells and ovarian cancer stem cells

There are a limited number of studies analyzing ovarian cancer stem cell properties. The goal of this study was to analyze the mechanical and migrational properties of ovarian cancer stem cells with the well-researched bulk ovarian cancer cells. Through the use of atomic force microscopy (AFM), the mechanical properties of both cell types were gathered. In preparation for AFM analysis, an optimal fixation method was developed and performed on the cells. AFM analysis provided mechanical properties for both cell types, including cellular stiffness, maximum adhesion force, surface area, and mean surface roughness. Cell proliferation and transwell migration assays were assessed to determine the aggressiveness of both cancer cell types. A clear trend between both cell types was expected for the mechanical and potential aggression properties. The data from both analyses were used to create a baseline for migration, proliferation, and mechanical properties of ovarian cancer stem cells and bulk ovarian cancer cells. Further studies will assess if these properties are impacted by chemotherapy exposure.

EFNA1 promotes the tumorigenesis and metastasis of cervical cancer by phosphorylation pathway and epithelial-mesenchymal transition

Cervical cancer (CC) is a common gynecological disease that seriously threatens women's health. This study aims to explore key genes and pathways related to CC prognosis through bioinformatics, providing new insights for further treatment of CC. CC patient data were analyzed from the public databases. The enrichment analyses explored the roles and pathways of CC-related differentially expressed genes (DEGs). A prognostic key gene was identified using Venn diagrams and subjected to survival analysis. Gene Set Enrichment Analysis (GSEA) was employed to investigate the potential pathways of key genes. Correlations between the key gene and clinical features were examined. The function of the key gene was validated through immunohistochemistry, flow cytometry, transwell, MTT, and Western blot assays in vitro and in vivo. Our research identified 2459 upregulated genes among DEGs between healthy and tumor cervical tissues. These DEGs were primarily enriched in the PI3K-AKT and MAPK pathways. Moreover, EFNA1 was recognized as a key prognostic gene in CC, with elevated expression compared to normal tissue. A negative correlation between EFNA1 levels and patient survival rates was corroborated by Kaplan-Meier analysis. Furthermore, EFNA1 expression correlated with the cancer stage and was linked to antigen presentation, folate synthesis, and IL-17 signaling. Knockdown of EFNA1 enhanced apoptosis and reduced migration, invasion, and proliferation in vitro and in vivo, inhibiting EMT and MAPK pathways. This study revealed the key signaling pathways in CC progression and identified EFNA1 as a crucial prognostic biomarker, potentially impacting CC treatment.

SOX2 promotes the glycolysis process to accelerate cervical cancer progression by regulating the expression of HK2

Cervical cancer is a major health burden in females worldwide, available studies indicated that sex-determining region Y-box 2 (SOX2) is closely related to the malignant phenotypes of multiple cancers including cervical cancer. However, the underlying mechanisms were blurred. A bioinformatics analysis was conducted to investigate the clinical correlation between SOX2 and cervical cancer. Transient transfection and lentivirus infection were utilized to achieve overexpression and knockdown of SOX2, respectively. The role of SOX2 in cervical cancer was confirmed by transwell and colony-forming assays. Immunoblot, dual-luciferase reporter, chromatin immunoprecipitation (ChIP), and biochemical experiments were employed. In addition, the xenograft models and immunohistochemistry (IHC) experiments were performed to validate the findings in vivo. The expression of SOX2 was significantly positively associated with the cell migration, invasion, and colony-forming abilities of cervical cancer cells. The following immunoblots revealed that the SOX2-induced malignant phenotypes might be related to the glycolysis process, since overexpressing SOX2 significantly promoted the hexokinase 2 (HK2) and glucose transporter-1 (GLUT1) expression, and increased the content of glucose and lactic acid. The further dual-luciferase reporter and ChIP experiments confirmed a binding relationship between SOX2 and HK2 promoter. More importantly, overexpressing SOX2 promoted tumor growth concomitant with a hyper-expression of HK2 and GLUT1 in xenograft tumor tissues, yet the treatment of glycolysis inhibitor significantly reversed those outcomes. SOX2 promotes the malignant progression of cervical cancer by facilitating glycolysis.