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(a) Squamous cell carcinoma (right) and adjacent mucosa with severe dysplasia (left) and a small area of mild dysplasia (∗). HE, orig. magnification 2x. (b) Invasive squamous carcinoma. HE, orig. magnification 20x. (c) Adjacent mucosa with severe dysplasia and a small area of mild dysplasia (∗). HE, orig. magnification 10x. (d) Expression of NANOG in carcinoma and in dysplastic epithelium in adjacent mucosa (∗). Immunohistochemistry, orig. magnification 2x. (e) Expression of NANOG in carcinoma, with cytoplasmic staining. Immunohistochemistry, orig. magnification, 20x. (f) Strong staining for NANOG in areas of severe dysplasia and weak staining in areas of mild dysplasia (∗). Immunohistochemistry, orig. magnification 10x.
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Background:
Results of previous studies suggest that NANOG may be an important prognostic biomarker in oral squamous cell carcinoma (OSCC), but there are contradictory results regarding NANOG expression patterns on mRNA and protein levels, and the mechanisms of its regulation are poorly understood. Our aim was to analyze the expression and diagnos...
Similar publications
Background:
Long noncoding RNA (lncRNA) is reported to be upregulated in many tumors. Although the expression of lncRNA in oral squamous cell carcinoma has been assessed, the association between lncRNA expression and prognosis or clinicopathological feature still remains controversial. Therefore, we conducted a meta-analysis to verify whether lncR...
Citations
... Higher expression of NANOG has been observed in various solid tumors of the breast, colon, lung, liver, and cervix. Elevated values of NANOG especially in cancer cells were described by previous research, which means that the NANOG gene is a critical member of cancer stem cells [14]. This study was done to evaluate the cancer stem cells in OSCC and OED using immunoexpression of NANOG. ...
... Sample size calculation was performed using the reported data [14,15] due to a lack of relevant literature. The tissue sections of oral leukoplakia with OED (n=28), OSCC (n=28), and normal oral mucosa (n=28) were taken from the archival blocks stored in the Department of Oral Pathology Department at PMS Dental College, Trivandrum, India. ...
... In the current study, NANOG protein expression was positive in 92.8% (n=26/28) of OSCC cases, all of which were primarily observed in the cytoplasm of tumor cells, whereas in germ cell tumors, a nuclear staining pattern was seen. Our results are similar to the study conducted by Grubelnik et al. who assessed both the protein and mRNA expressions of NANOG in 30 samples of OSCC and found that 27 cases exhibited positive staining mainly in the cytoplasm of tumor cells [14]. ...
Background: Squamous cell carcinoma of the oral cavity may show precursor lesions, termed as potentially malignant disorders, of which leukoplakia is the most frequent one. Oral leukoplakia is a clinical diagnosis for which the histological diagnosis may be either hyperplasia or oral epithelial dysplasia (OED) and sometimes even oral squamous cell carcinoma (OSCC). Cancer stem cells (CSCs), identified in various tumors, are a specific group of cells that exhibit the properties of self-renewal and differentiation. Among the various biomarkers that identify CSCs, the transcription factor NANOG is considered to be a significant one.
Aim: In this study, we intend to identify and compare the immunohistochemical expression of NANOG in OSCC, OED, and normal oral mucosa.
Methodology: Tissue blocks of OSCC (n=28), OED (n=28), and normal oral mucosa (n=28) were used in this study. Specimens were immunohistochemically analyzed for NANOG expression. The results were statistically analyzed using one-way ANOVA, Games-Howell post hoc, and Student t-test. Statistical Product and Service Solutions (SPSS, version 21; IBM SPSS Statistics for Windows, Armonk, NY) software was used for performing the statistical analysis, and the level of significance was set as 0.05.
Observations: NANOG expression was higher in OSCC when compared to oral dysplasias and normal oral mucosa, in decreasing order. A significantly higher histo-score and labeling index score were observed in OSCC and oral dysplasias compared to normal oral mucosa (p=<0.001).
Conclusion: The expression levels of NANOG were positively correlated with disease progression in OSCC, implicating that NANOG can be used as a surrogate marker of oral oncogenesis and prognosis. Therefore, decoding the molecular mechanisms of NANOG regulation in the progression of cancer helps in developing new therapeutic strategies for oral cancer.
... In support of a role as favorable prognostic biomarker, it has been reported that SOX2 is overexpressed during the early stages of OSCC and that this predicts for reduced local recurrence [42]. On the contrary, other studies have demonstrated that SOX2 overexpression promotes EMT and lymph node metastasis [42][43][44][45][46][47] while SOX2 knockdown inhibits HNSCC cell self-renewal and chemoresistance [34]. A recent retrospective study also highlights that SOX2 is overexpressed in pre-malignant oral disorders and that this predicts for oral cancer evolution [42,48]. ...
Emerging evidence shows that oral squamous cell carcinoma (OSCC) invasiveness can be attributed to a small subpopulation of cancer stem cells (CSCs) in the bulk of the tumor. However, the presence of CSCs in the OSCC close resection margins is still poorly unexplored. Here, we found that BMI1 , CD44 , SOX2 , OCT4 , UBE2C , CXCR4 CSCs marker genes are significantly upregulated, while IGF1-R , KLF4 , ALDH1A1 , CD133 , FAM3C are downregulated in the tumor core vs healthy mucosa of 24 patients with OSCC. Among these, SOX2 appears also upregulated in the tumor close margin vs healthy mucosa and this significantly correlates with tumor size and lymph node compromise. In vitro analyses in CAL27 and SCC15 tongue squamous cell carcinoma cell lines, show that SOX2 transient knockdown i) promotes the mesenchymal-to-epithelial transition, ii) smooths the invasiveness, iii) attenuates the 3D tumor sphere-forming capacity, and iv) partially increases the sensitivity to cisplatin treatment. Overall, our study highlights that the OSCC close margins can retain CSC-specific markers. Notably, SOX2 may represent a useful CSCs marker to predict a more aggressive phenotype and a suitable target to prevent local invasiveness.
... Their result emphasized the pivotal role of protein regulators (like NOTCH1) and microRNAs (such as miR-34a) more than promoter methylation and copy number variation of NANOG. [31] Although some previous studies emphasized on tumor suppressor role of NOTCH1 in OSCC, a novel mutation known as C1133Y was detected in the Chinese population that truncated protein cannot present at the cell surface. In this way, C1133Y mutated NOTCH1 activated the EGFR-PI3K/ AKT and improved proliferation and invasion in OSCC. ...
Background
Oral squamous cell carcinoma (OSCC) is the most common type of oral cancer with heterogeneous molecular pathogenesis. Oral lichen planus (OLP) is demonstrated potentially can transfer to OSCC malignant lesions. Unfortunately, there are no definitive prognostic and predictive biomarkers for the clinical management of OSCC patients. The present research is the first study that compared an oral premalignant lesion such as OLP to malignant lesions like OSCC for NOTCH1 expression levels to better understand its oncogenic or tumor suppressive role.
Materials and Methods
In this cross-sectional study, mRNA expression of NOTCH1 was evaluated by quantitative polymerase chain reaction in 65 tissue-embedded Paraffin-Block samples, including 32 OSCC and 33 OLP. Furthermore, we collected demographic information and pathological data, including tumor stage and grade. The association between NOTCH1 and GAPDH gene expressions was determined by Chi-squared, Spearman, and Mann–Whitney tests. A P < 0.05 was considered statistically significant for all statistical analyses.
Results
Comparison of OSCC and OLP groups showed a statistically significant difference between the quantitative expression of the NOTCH1 gene (P < 0.001). Qualitative gene expression was divided into low expression and high expression. Both study groups demonstrated a statistically significant gene expression difference (P < 0.001). There was a statistically significant difference between age and NOTCH1 expression in the OLP group (P = 0.036). There was no correlation between NOTCH1 expression and age, gender, tumor grade, and stage.
Conclusion
Since the OSCC is a malignant lesion and the OLP showed the possible nature of malignancy transformation, we can consider the NOTCH1 as a biomarker for the assessment of the tumorigenesis process with a definition of a standard threshold for potentially malignant lesions and malignant OSCC tumors.
... According to Grubelnik et al. Nanog and OCT4 genes were overexpressed in cancers with lymph node metastasis compared to cases without metastases [44]. In our study, the qPCR experiment showed that all OLs and OSCCs revealed SOX2 and OCT3/4genes but failed to establish any statistically significant correlation among them. ...
Introduction: Cancer stem cells (CSCs) are incriminated for initiating the process of carcinogenesis either de novo or through the transformation of oral potentially malignant disorders (OPMDs) to oral squamous cell carcinoma (OSCC). The aim of this study was to detect the expression of embryonic-type CSC markers OCT3/4 and SOX2 in OSCCs and oral leukoplakias (OLs), the most common of OPMDs.
Materials and methods: The study type is experimental, and the study design is characterized as semiquantitative research, which belongs to the branch of experimental research. The experiment was conducted in the Department of Oral Medicine/Pathology, School of Dentistry, Aristotle University of Thessaloniki, Greece. This study focuses on the semiquantitative immunohistochemical (IHC) pattern of expression of CSCs protein-biomarkers SOX2 and OCT3/4, in paraffin embedded samples of 21 OSCCs of different grades of differentiation and 30 cases of OLs with different grades of dysplasia, compared to five cases of normal oral mucosa in both terms of cells’ stain positivity and intensity. Statistical analysis was performed through SPSS 2017 Pearson Chi-square and the significance level was set at 0.05 (p=0.05). The expression of the respective genes of SOX2 and OCT3/4 was studied through quantitative polymerase chain reaction (qPCR), in paraffin-embedded samples of 12 cases of OLs with mild/non dysplasia and 19 cases moderately/poorly differentiated OSCCs(n=19) and five normal mucosa using the Independent Paired T-test.
Results: The genes SOX2 and Oct3/4 were expressed in all examined cases although no statistically significant correlations among normal, OL and OSCC, were established. A nuclear/membrane staining of OCT3/4 was noticed only in three out of 21 OSCCs but in none of OLs or normal cases (without statistical significance). A characteristic nuclear staining of SOX2 was noticed in the majority of the samples, mostly in the basal and parabasal layers of the epithelium. SOX2 was significantly detected in the OSCCs group (strong positivity in 17/21) than in the OL group (30 cases, mostly mildly stained) (p-value=0.007), and the normal oral epithelium (mild stained, p=0.065). Furthermore, SOX2 was overexpressed in well differentiated OSCCs group (5/OSCCs, strongly stained) rather than in mildly dysplastic and non-dysplastic OLs samples (14/OLs, mildly stained) (p-value =0.035).
Conclusion:The characteristic expression of SOX2 but not of OCT3/4 in OLs' and OSCCs’ lesions suggests the presence of neoplastic cells with certain CSC characteristics whose implication in the early stages of oral tumorigenesis could be further evaluated. The clinical use of SOX2, as prognostic factor, requires further experimental evaluation in larger number of samples.
... There is not any bibliography on qPCR implementation in OL and OSCC cases for the study of CD147. The only reference on qPCR and cancer stem cells reported that the embryonic stem cells' markers Nanog and OCT4 were overexpressed in cancers with lymph node metastasis than in cancers without lymph node metastasis [46]. The limitations of our study included the lack of follow-ups of the patients from whom the tissue specimens were derived, the lack of tumor-node-metastasis (TNM) classification and of the human papillomavirus (HPV) status regarding the OSCC included, and the lack of study of additional target molecules such as VEGF. ...
Abstract
Objectives
Cancer stem cells (CSCs) are responsible for initiating the process of carcinogenesis de novo, as well as through the transformation of oral potential malignant disorders (OPMDs) to oral squamous cell carcinoma (OSCC). The aim of our study was to detect the expression of stemness-type CSC marker CD147 in oral leukoplakias (OLs), the most common OPMD, and OSCCs as well.
Materials and methods
This study focuses on the semiquantitative immunohistochemical pattern of the expression of the CSC protein biomarker CD147 in paraffin-embedded samples of 20 OSCCs of different grades of differentiation and 30 cases of OLs without or with different grades of dysplasia, compared to the normal oral epithelium in terms of cells’ stain positivity. Statistical analysis was performed through Statistical Package for Social Sciences (SPSS) version 25.0 (IBM SPSS Statistics, Armonk, NY) with Pearson chi-square test, and the significance level was set at 0.05 (p=0.05). In addition, the study clarified the expression of the respective gene of CD147 through quantitative polymerase chain (qPCR), in paraffin-embedded samples of the two extreme graduations: OLs of mildly dysplastic or non-dysplastic cases (n=10 cases) and OSCCs of moderately/poorly differentiated cases (n=17). Statistical analysis was then performed through SPSS version 25.0 with an independent paired t-test, and the significance level was set at 0.05 (p=0.05).
Results
The gene CD147 was expressed in all cases, although no statistically significant correlations were established. Regarding its protein products, the characteristic membranous staining of CD147 was noticed in the majority of the samples, mostly in the basal and parabasal layers of the epithelium. CD147 was upregulated significantly in the moderately and severely dysplastic OLs than in the mildly dysplastic and non-dysplastic OLs (p=0.008). Also, CD147 was upregulated significantly in the mildly dysplastic and non-dysplastic OLs than in the normal oral epithelium (p=0.012).
Discussion
The characteristic expression of CD147 in OLs and OSCCs’ lesions suggests the presence of stemlike cancer cells, illustrating an underlying effect on the early stages of oral dysplasia, in the OL stage. The clinical application of CD147 as prognostic factor requires the experimental evaluation in larger number of samples.
Conclusion
Stem cells play an important role in the process of carcinogenesis. A major goal in cancer research is the identification of specific biomarkers for the detection of cancer stem cells. CD147 is considered as an innovative stem cell marker. Our findings in oral mucosal potentially malignant disorders showed that CD147 is expressed more intensely in parallel with the progression of the grade of dysplasia in OL. On the other hand, in oral squamous cell carcinoma, CD147 expression remains stable regardless of the degree of differentiation.
... POU5F1, SOX2, and NANOG are important for the tumorigenesis of skin, oral squamous, esophageal, lung, and colon cancer [193,194]. SOX2 contributes to the development of brain tumors [195]. NANOG is also an important mediator of cancer induction [196]. ...
In testicular germ cell tumor type II (TGCT), a seminoma subtype expresses an induced pluripotent stem cell (iPSC) panel with four upregulated genes, OCT4/POU5F1, SOX17, KLF4, and MYC, and embryonal carcinoma (EC) has four upregulated genes, OCT4/POU5F1, SOX2, LIN28, and NANOG. The EC panel can reprogram cells into iPSC, and both iPSC and EC can differentiate into teratoma. This review summarizes the literature on epigenetic regulation of the genes. Epigenetic mechanisms, such as methylations of cytosines on the DNA string and methylations and acetylations of histone 3 lysines, regulate expression of these driver genes between the TGCT subtypes. In TGCT, the driver genes contribute to well-known clinical characteristics and the driver genes are also important for aggressive subtypes of many other malignancies. In conclusion, epigenetic regulation of the driver genes are important for TGCT and for oncology in general.
... The high expression of AGR2 has been previously associated with the expression of Nestin, CD133 and SOX2 in high-grade meningioma tissues [39]. AGR2 expression was also shown to have a strong positive correlation with nanog homeobox (NANOG) in oral squamous cell carcinoma [73]. The effect of high GRP78 expression in glioblastoma-CSCs is still being investigated. ...
Background
Glioblastomas (GBs) are characterised as one of the most aggressive primary central nervous system tumours (CNSTs). Single-cell sequencing analysis identified the presence of a highly heterogeneous population of cancer stem cells (CSCs). The proteins anterior gradient homologue 2 (AGR2) and glucose-regulated protein 78 (GRP78) are known to play critical roles in regulating unfolded protein response (UPR) machinery. The UPR machinery influences cell survival, migration, invasion and drug resistance. Hence, we investigated the role of AGR2 in drug-resistant recurrent glioblastoma cells.
Methods
Immunofluorescence, biological assessments and whole exome sequencing analyses were completed under in situ and in vitro conditions. Cells were treated with CNSTs clinical/preclinical drugs taxol, cisplatin, irinotecan, MCK8866, etoposide, and temozolomide, then resistant cells were analysed for the expression of AGR2. AGR2 was repressed using single and double siRNA transfections and combined with either temozolomide or irinotecan.
Results
Genomic and biological characterisations of the AGR2-expressed Jed66_GB and Jed41_GB recurrent glioblastoma tissues and cell lines showed features consistent with glioblastoma. Immunofluorescence data indicated that AGR2 co-localised with the UPR marker GRP78 in both the tissue and their corresponding primary cell lines. AGR2 and GRP78 were highly expressed in glioblastoma CSCs. Following treatment with the aforementioned drugs, all drug-surviving cells showed high expression of AGR2. Prolonged siRNA repression of a particular region in AGR2 exon 2 reduced AGR2 protein expression and led to lower cell densities in both cell lines. Co-treatments using AGR2 exon 2B siRNA in conjunction with temozolomide or irinotecan had partially synergistic effects. The slight reduction of AGR2 expression increased nuclear Caspase-3 activation in both cell lines and caused multinucleation in the Jed66_GB cell line.
Conclusions
AGR2 is highly expressed in UPR-active CSCs and drug-resistant GB cells, and its repression leads to apoptosis, via multiple pathways.
... However, protein and mRNA expression levels sometimes did not match. At the mRNA level, it was positively correlated with other cancer malignancy-associated factors: OCT4, SOX2, NOTCH1, AGR2, and KLF4 [29]. ...
The use of extracellular vesicle (EV)-based vaccines is a strategically promising way to prevent cancer metastasis. The effective roles of immune cell-derived EVs have been well understood in the literature. In the present paper, we focus on cancer cell-derived EVs to enforce, more thoroughly, the use of EV-based vaccines against unexpected malignant cells that might appear in poor prognostic patients. As a model of such a cancer cell with high malignancy, Nanog-overexpressing melanoma cell lines were developed. As expected, Nanog overexpression enhanced the metastatic potential of melanomas. Against our expectations, a fantastic finding was obtained that determined that EVs derived from Nanog-overexpressing melanomas exhibited a metastasis-suppressive effect. This is considered to be a novel role for Nanog in regulating the property of cancer cell-derived EVs. Stimulated by this result, the review of Nanog’s roles in various cancer cells and their EVs has been updated once again. Although there was no other case presenting a similar contribution by Nanog, only one case suggested that NANOG and SOX might be better prognosis markers in head and neck squamous cell carcinomas. This review clarifies the varieties of Nanog-dependent phenomena and the relevant signaling factors. The information summarized in this study is, thus, suggestive enough to generate novel ideas for the construction of an EV-based versatile vaccine platform against cancer metastasis.
... CSCs constitute a subpopulation of tumor cells endowed with properties such as self-renewal and long-term clonal persistence [7], maintained in specific niches within the tumor in which the interaction with the TME is crucial [8]. In turn, CSCs bear stemness properties supported by gene master regulators, such as NANOG, SOX2 and OCT4 [9], which have been implicated in oral carcinogenesis, poor differentiation and poor prognosis [10][11][12][13][14][15][16][17]. Accumulating evidence indicates that these transcription factors correlate with Nestin expression [18]. ...
... It has been shown that PDPN regulates stem cells in normal and tumor tissues [22], and also reported as a valuable biomarker for cancer risk assessment of malignant transformation in oral leukoplakia [23]. NANOG is also a pluripotency/CSC regulatory factor, found to be widely detected in multiple cancers, enriched in tumor cells that exhibit stem cell-like properties and with important prognostic implications in several cancer types, including OSCC [11,13]. The association between TILs and patient survival has been described in various types of cancer [1,[24][25][26][27][28][29][30][31][32][33]. ...
... NANOG not only contributes to the regulation of pluripotency in stratified epithelia [80]; it also mediates tumor cell proliferation, epithelial-mesenchymal transition and escape from immune system [78]. NANOG expression in OSCC is regulated by SOX2, OCT4, KLF4, AGR2, NOTCH1 and miR-34a [13]. However, OCT4 expression was not detected in any tumor in our OSCC cohort. ...
This study investigates the relevance of tumor-infiltrating lymphocytes (TILs) in oral squamous cell carcinoma (OSCC). Immunohistochemical analysis of stromal/tumoral CD4+, CD8+ and FOXP3+ TILs is performed in 125 OSCC patients. Potential relationships with the expression of tumoral PD-L1 and cancer stem cell (CSC) markers (NANOG, SOX2, OCT4, Nestin and Podoplanin (PDPN)) are assessed. CD4+ and CD8+ TILs are significantly associated with smoking and alcohol habits. CD4+ and CD8+ TILs show an inverse relationship with NANOG and SOX2 expression, and FOXP3+ TILs is significantly correlated with Nestin and PDPN expression. High infiltration of CD4+ and CD8+ TILs and a high tumoral CD8+/FOXP3+ ratio are significantly associated with tumors harboring positive PD-L1 expression. Infiltration of stromal/tumoral FOXP3+ TILs and a low stromal CD8+/FOXP3+ ratio are significantly associated with better disease-specific survival. Multivariate analysis reveals that the stromal CD8+/FOXP3+ TILs ratio is a significant independent prognostic factor. Regarding OSCC patient survival, the CD8+/FOXP3+ TILs ratio is an independent prognostic factor. TILs may act as biomarkers and potential therapeutic targets for OSCC.
... As well, a positive feedback loop comprising HLF and the pluripotency factors SOX2 and OCT4 may interplays with NANOG to regulate stemness. Recent studies have shown that the pluripotency factors SOX2 and OCT4 and NANOG are involved in its regulation during OSCC development at least to certain degree [21][22][23]. Therefore, it may be hypothesised that HLF plays a major role in the development of HNSCC. ...
Background
Head and neck squamous cell carcinoma (HNSCC) is one of the most frequent cancers worldwide, with an increasing incidence. However, the underlying molecular mechanisms of HNSCC are poorly understood.
Method
In this work, 5 original datasets (GSE23558, GSE13601, GSE30784, GSE9844, GSE78060) of Head and neck squamous cell carcinoma (HNSCC) were selected from Gene Expression Omnibus (GEO) database. To identify differentially expressed genes (DEGs) in HNSCC and adjacent tissues. The common DEGs were acquired by Venn diagram. The sensitivity and specificity of HLF were determined by Receiver operating characteristic curves (ROC). Then, In order to further confirm the relationship between HLF and HNSCC patient’s prognosis, the expression and survival analysis of HLF was performed by Gene Expression Profiling Interactive Analysis (GEPIA), Cell culture, reverse transcription polymerase chain reaction (RT-PCR), western blotting and immunohistochemical staining.
Results
Seventeen DEGs were screened from five sets of HNSCC functional gene expression series in GEO datasets. The low expression of HLF was indicated might be correlated with poor prognosis of HNSCC patients based on the bioinformatics analysis. According to the results of Cell culture, RT-PCR, western blotting, immunohistochemical staining, it was confirmed that the low level of HLF expression correlated with poor prognosis of HNSCC patients.
Conclusion
The study effectively revealed useful information about the relationship of the low level of HLF expression and HNSCC. In summary, we identified HLF as a potential prognostic biomarker and therapeutic target for HNSCC.
