Erk1/2-Dependent HNSCC Cell Susceptibility to Erastin-Induced Ferroptosis.

Unfavorable clinical outcomes mean that cancer researchers must attempt to develop novel therapeutic strategies to overcome therapeutic resistance in patients with HNSCC. Recently, ferroptosis was shown to be a promising pathway possessing druggable targets, such as xCT (SLC7A11). Unfortunately, little is known about the molecular mechanisms underlying the susceptibility of HNSCC cells to ferroptosis. The goal of this study was to determine whether HNSCC cells with activated Erk1/2 are vulnerable to ferroptosis induction. Our results have shown that xCT (SLC7A11) was overexpressed in malignant tissues obtained from the patients with HNSCC, whereas normal mucosa demonstrated weak expression of the protein. In order to investigate the role of Erk1/2 in the decrease in cell viability caused by erastin, xCT-overexpressing FaDu and SCC25 HNSCC cells were used. The ravoxertinib-dependent inhibition of Erk1/2 signaling led to the decrease in erastin efficacy due to the effect on ROS production and the upregulation of ROS scavengers SOD1 and SOD2, resulting in repressed lipid peroxidation. Therefore, it was concluded that the erastin-dependent activation of ferroptosis seems to be a promising approach which can be further developed as an additional strategy for the treatment of HNSCC. As ferroptosis induction via erastin is strongly dependent on the expression of Erk1/2, this MAP kinase can be considered as a predictor for cancer cells' response to erastin.

Interplay between Partial EMT and Cisplatin Resistance as the Drivers for Recurrence in HNSCC.

This study aims to investigate the role of partial epithelial to mesenchymal transition (pEMT)-related proteins in modulating Cisplatin resistance in head and neck squamous cell carcinoma (HNSCC). SCC-25 cells were pre-treated with TGF-beta1 followed by transient Krüppel-like Factor 4 (KLF4)-overexpression and Cisplatin treatment. Cell growth, cell morphological changes and cell migration were assessed using Juli BR live cell video-microscopy. In addition, Ki-67 and Slug immunostaining and follow-up image cytometric analysis of primary and recurrent HNSCC tumors were performed to evaluate the proliferation index (PI) and the EMT-like phenotype. We observed that proliferating and Slug-positive tumor cells expand after therapy in HNSCC. Subsequently, protein analysis revealed the stabilization of Slug, upregulation of Vimentin and phospho-p38 (p-p38) in Cisplatin-resistant SCC-25 cells. Moreover, KLF4-overexpression contributed to Cisplatin sensitivity by reduction of Slug at the protein level. This work strongly suggests that an pEMT-like pathway is activated in recurrent and Cisplatin-resistant HNSCC. Finally, stable KLF4-overexpression might sensitize HNSCC tumor cells for Cisplatin treatment.

EMT-related transcription factors and protein stabilization mechanisms involvement in cadherin switch of head and neck squamous cell carcinoma.

Epithelial to mesenchymal transition (EMT) describes a process where epithelial tumor cells acquire mesenchymal characteristics. EMT often correlates with invasion and an increased cell migration potential by losing cellular polarity and cell-cell junctions. It is mainly induced by tumor-microenvironment factors, such as TGF-beta 1 and IL-6, which activate the increased expression of the EMT-transcription factor (TF) Slug. We previously reported the Slug/Krüppel-like factor 4 (KLF4) switch in EMT in HNSCC, and found, that in human papilloma virus (HPV)-negative HNSCC Slug gene expression was significant higher represented, than in HPV-positive HNSCC. The purpose of this study was to investigate the impact of KLF4 and Slug on the regulation of the cadherin switch and on the EMT phenotype. Gene expression of KLF4 positive correlated with E-cadherin in 71 head and neck squamous cell carcinoma (HNSCC) patient tissue samples, which we also confirmed by the investigation of the Cancer Genome Atlas database (TCGA). HPV-transcripts contributed to stabilization of KLF4 at protein level, and simultaneously upregulated E-cadherin. Furthermore, ectopic KLF4 overexpression was associated with epithelial gene expression by induction of E-cadherin, ß-catenin and 70-kDa heat shock protein (HSP-70). The presence of HSP-70 ensures the membranous localization of E-cadherin, therefore, the ability of cells to form cadherin/catenin complexes and cellular linkages. In conclusion, KLF4 is a major regulator of the epithelial cadherin-adhesion in HNSCC.

KLF4, Slug and EMT in Head and Neck Squamous Cell Carcinoma.

Epithelial to mesenchymal transition (EMT) is clinically relevant in head and neck squamous cell carcinoma (HNSCC). We hypothesized that EMT-transcription factors (EMT-TFs) and an anti-EMT factor, Krüppel-like-factor-4 (KLF4) regulate EMT in HNSCC. Ten control mucosa and 37 HNSCC tissue samples and three HNSCC cell lines were included for investigation of EMT-TFs, KLF4 and vimentin at mRNA and protein levels. Slug gene expression was significantly higher, whereas, KLF4 gene expression was significantly lower in HNSCC than in normal mucosa. In the majority of HNSCC samples, there was a significant negative correlation between KLF4 and Slug gene expression. Slug gene expression was significantly higher in human papilloma virus (HPV) negative HNSCC, and in tumor samples with irregular p53 gene sequence. Transforming-growth-factor-beta-1 (TGF- ß1) contributed to downregulation of KLF4 and upregulation of Slug. Two possible regulatory pathways could be suggested: (1) EMT-factors induced pathway, where TGF-ß1 induced Slug together with vimentin, and KLF4 was down regulated at the same time; (2) p53 mutations contributed to upregulation and stabilization of Slug, where also KLF4 could co-exist with EMT-TFs.

Characterization of epithelial cells, connective tissue cells and immune cells in human upper airway mucosa by immunofluorescence multichannel image cytometry: a pilot study.

Epithelial, connective tissue and immune cells contribute in various ways to the pathophysiology of chronic rhinosinusitis (CRS). However, data of their distribution in upper airway mucosa are sparse. We aimed to provide quantitative, purely informative data on the distribution of these cell lineages and their coexpression patterns, which might help identifying, e.g., cells in the epithelium undergoing through epithelial-mesenchymal transition (EMT). For this purpose, we used immunofluorescence multichannel image cytometry (IMIC). We examined fixed paraffin-embedded tissue samples (FFPE) of six patients with chronic rhinosinusitis (CRS) and of three patients without CRS (controls). The direct-conjugated antibodies pancytokeratin, vimentin and CD45/CD18 were used for coexpression analysis in epithelial layer and lamina propria. Image acquisition and analysis were performed with TissueFAXS and StrataQuest, respectively. To distinguish positive from negative expression, a ratio between cell-specific immunostaining intensity and background was developed. Isotype controls were used as negative controls. Per patient, a 4.5-mm2 tissue area was scanned and a median of 14,875 cells was recognized. The most common cell types were cytokeratin-single-positive (26%), vimentin-single-positive (13%) and CD45/CD18-single-positive with CD45/CD18-vimentin-double-positive cells (29%). In the patients with CRS, CD45/CD18-single-positive cells were 3-6 times higher compared to the control patients. In the epithelial layer, cytokeratin-vimentin-double-positive EMT cells were observed 3-5 times higher in the patients with CRS than in the control patients. This study provided quantitative data for the distribution of crucial cell types in CRS. Future studies may focus on the distribution and coexpression patterns of different immune cells in CRS or even cancer tissue.

HPV-Induced Oropharyngeal Cancer and the Role of the E7 Oncoprotein Detection via Brush Test.

Background: High risk human papillomavirus (hr-HPV)-associated oropharyngeal cancers (OPCs) are characterized by significantly better therapy responses. In order to implement a de-escalated treatment strategy for this tumor entity, it is highly crucial to accurately distinguish HPV-associated OPCs from non-HPV-associated ones. Methods: In this prospective study, 56 patients with histologically confirmed OPC were evaluated. A commercially available sandwich ELISA test system was used for the detection of hr-HPV E7 oncoprotein targeting the genotypes 16, 18 and 45. Results were presented as optical density. Positivity for HPV DNA and p16 immunohistochemistry (IHC) was taken as the reference method. Results: E7 positivity was significantly associated with the reference method (p = 0.048). The sensitivity, specificity, positive predictive value and negative predictive value for the E7 oncoptotein was 60.9% (95% CI 38.5 to 80.3%), 66.7% (95% CI 46% to 83.5%), 64.2% (95% CI 49.4 to 77.4%) and 63.01% (95% CI 48.9-75.2%), respectively, for the cutoff provided by the manufacturer. Conclusions: We found a significant association between E7 oncoprotein detection and the currently used combination. We believe that the use of the ELISA based E7 antigen test could be a valuable addition in cases of ambiguous findings and may be used in combination with other techniques to distinguish between HPV-driven and non-HPV-driven OPCs. However, the low sensitivity of the assay coupled with the small sample size in our study may represent a limitation. We recommend that future larger studies elucidate the diagnostic value of the E7 brush test.

H3K27me3 expression and methylation status in histological variants of malignant peripheral nerve sheath tumours.

Diagnosing MPNST can be challenging, but genetic alterations recently identified in polycomb repressive complex 2 (PRC2) core component genes, EED and SUZ12, resulting in global loss of the histone 3 lysine 27 trimethylation (H3K27me3) epigenetic mark, represent drivers of malignancy and a valuable diagnostic tool. However, the reported loss of H3K27me3 expression ranges from 35% to 84%. We show that advances in molecular pathology now allow many MPNST mimics to be classified confidently. We confirm that MPNSTs harbouring mutations in PRC2 core components are associated with loss of H3K27me3 expression; whole-genome doubling was detected in 68%, and SSTR2 was amplified in 32% of MPNSTs. We demonstrate that loss of H3K27me3 expression occurs overall in 38% of MPNSTs, but is lost in 76% of histologically classical cases, whereas loss was detected in only 23% cases with heterologous elements and 14% where the diagnosis could not be provided on morphology alone. H3K27me3 loss is rarely seen in other high-grade sarcomas and was not found to be associated with an inferior outcome in MPNST. We show that DNA methylation profiling distinguishes MPNST from its histological mimics, was unrelated to anatomical site, and formed two main clusters, MeGroups 4 and 5. MeGroup 4 represents classical MPNSTs lacking H3K27me3 expression in the majority of cases, whereas MeGroup 5 comprises MPNSTs exhibiting non-classical histology and expressing H3K27me3 and cluster with undifferentiated sarcomas. The two MeGroups are distinguished by differentially methylated PRC2-associated genes, the majority of which are hypermethylated in the promoter regions in MeGroup 4, indicating that the PRC2 target genes are not expressed in these tumours. The methylation profiles of MPNSTs with retention of H3K27me3 in MeGroups 4 and 5 are independent of mutations in PRC2 core components and the driver(s) in these groups remain to be identified. Our results open new avenues of investigation. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.

Epithelial to Mesenchymal Transition: A Mechanism that Fuels Cancer Radio/Chemoresistance.

Epithelial to mesenchymal transition (EMT) contributes to tumor progression, cancer cell invasion, and therapy resistance. EMT is regulated by transcription factors such as the protein products of the SNAI gene family, which inhibits the expression of epithelial genes. Several signaling pathways, such as TGF-beta1, IL-6, Akt, and Erk1/2, trigger EMT responses. Besides regulatory transcription factors, RNA molecules without protein translation, micro RNAs, and long non-coding RNAs also assist in the initialization of the EMT gene cluster. A challenging novel aspect of EMT research is the investigation of the interplay between tumor microenvironments and EMT. Several microenvironmental factors, including fibroblasts and myofibroblasts, as well as inflammatory, immune, and endothelial cells, induce EMT in tumor cells. EMT tumor cells change their adverse microenvironment into a tumor friendly neighborhood, loaded with stromal regulatory T cells, exhausted CD8+ T cells, and M2 (protumor) macrophages. Several EMT inhibitory mechanisms are instrumental in reversing EMT or targeting EMT cells. Currently, these mechanisms are also significant for clinical use.

Frost Survival Mechanism of Vegetative Buds in Temperate Trees: Deep Supercooling and Extraorgan Freezing vs. Ice Tolerance.

In temperate climates, overwintering buds of trees are often less cold hardy than adjoining stem tissues or evergreen leaves. However, data are scarce regarding the freezing resistance (FR) of buds and the underlying functional frost survival mechanism that in case of supercooling can restrict the geographic distribution. Twigs of 37 temperate woody species were sampled in midwinter 2016 in the Austrian Inn valley. After assessment of FR, infrared-video-thermography and cryo-microscopy were used to study the freezing pattern in and around overwintering vegetative buds. Only in four species, after controlled ice nucleation in the stem (-1.6 ± 0.9°C) ice was observed to immediately invade the bud. These buds tolerated extracellular ice and were the most freezing resistant (-61.8°C mean LT50). In all other species (33), the buds remained supercooled and free of ice, despite a frozen stem. A structural ice barrier prevents ice penetration. Extraorgan ice masses grew in the stem and scales but in 50% of the species between premature supercooled leaves. Two types of supercooled buds were observed: in temporary supercooling buds (14 species) ice spontaneously nucleated at -20.5 ± 4,6°C. This freezing process appeared to be intracellular as it matched the bud killing temperature (-22.8°C mean LT50). This response rendered temporarily supercooled buds as least cold hardy. In 19 species, the buds remained persistently supercooled down to below the killing temperature without indication for the cause of damage. Although having a moderate midwinter FR of -31.6°C (LT50), some species within this group attained a FR similar to ice tolerant buds. The present study represents the first comprehensive overview of frost survival mechanisms of vegetative buds of temperate trees. Except for four species that were ice tolerant, the majority of buds survive in a supercooled state, remaining free of ice. In 50% of species, extraorgan ice masses harmlessly grew between premature supercooled leaves. Despite exposure to the same environmental demand, midwinter FR of buds varied intra-specifically between -17.0 and -90.0°C. Particularly, species, whose buds are killed after temporary supercooling, have a lower maximum FR, which limits their geographic distribution.