Nuclear factor IB is downregulated in vulvar squamous cell carcinoma (VSCC): Unravelling differentially expressed genes in VSCC through gene expression dataset analysis
- Authors:
- Published online on: March 16, 2021 https://doi.org/10.3892/ol.2021.12642
- Article Number: 381
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Copyright: © Dasgupta et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
Abstract
Introduction
Vulvar squamous cell carcinoma (VSCC) constitutes 90% of all vulvar malignancies, and its incidence has risen over the past decades (1,2). Approximately 25% of VSCCs arise in association with a human papillomavirus (HPV)-infection, via the precursor lesion, high grade squamous intraepithelial lesion (HSIL) (3). The majority (75%) of VSCCs, however, is postulated to develop on the background of chronic dermatoses, via the precursor lesion, differentiated vulvar intraepithelial neoplasia (dVIN) (3).
The dual pathogenesis of VSCC has been recognized several years ago, however, molecular mechanisms of the carcinogenesis have not been well characterized (4). This is largely because the genomic profiles of VSCC or its precursor lesions have been investigated in only a few studies so far (1,5–11). These studies identified somatic mutations of TP53 to be the pivotal oncogenic driver of HPV-independent VSCC, and also detected genomic alterations of PIK3CA, HRAS, or FGFR3 in both subtypes of VSCC (7–9,11). Nevertheless, limited sample sizes and dissimilar methodologies of these studies have prevented significant advancement of knowledge of VSCC carcinogenesis (4).
A better understanding of the molecular pathways involved in VSCC carcinogenesis can enable identification of biomarkers that may be used to improve the diagnosis, for prognostic stratification, or as targets for precision treatment. Currently, the mainstay of VSCC treatment is surgical excision, which is often associated with post-operative morbidities due to the anatomical complexity of the vulvar region. Discovering novel biomarkers for targeted treatment may help improve personalization of treatment for patients with VSCC.
A key method for discovering candidate biomarkers is through identifying genes that are differentially expressed in cancer tissue and normal tissue (12). To this end, we analyzed datasets of gene expression microarray on VSCC and normal vulvar tissue, from two independent studies, using the latest bioinformatics tools. We further investigated the expression of some of the differentially expressed genes (DEGs) identified thereby, by performing immunohistochemistry (IHC) on VSCC, HSIL, dVIN, and normal vulvar tissue.
Materials and methods
Identification and analysis of datasets
A publicly available dataset (GSE38228) was identified and downloaded from gene expression omnibus (GEO) (13). This dataset consists of VSCCs (n=14) and normal vulvar tissues (n=5), for which gene expression microarray had been performed using the gene-chip platform Illumina HumanHT-12 V4.0. A 2nd dataset was obtained from a study previously conducted by researchers at our center. This dataset consists of VSCCs (n=5), for which gene expression microarray was performed using the gene-chip platform Affymetrix HG U133 Plus 2.0.
The datasets were imported into OmniViz (version 6.1.13.0, BioWisdomLtd.). Statistical analysis of microarrays (SAM) was performed to identify DEGs using the following cutoff-values-a false discovery rate (FDR) of ≤0.01 and a fold change of 1.5. P-value <0.05 was considered as statistically significant. Functional annotations of the SAM results were done using Ingenuity Pathway Analysis (IPA, Qiagen, Inc.). Expression levels of p16 (CDKN2A), which is known to be overexpressed in HPV-related VSCC, were used to distinguish the samples as HPV-related or HPV-independent VSCC. For both subtypes of VSCC, DEGs that were upregulated or downregulated in both datasets with statistical significance were identified. The Database for Annotation, Visualization and Integrated Discovery (DAVID; version 6.8) was used to identify the most significantly enriched functional genes (14,15). Gene ontology (GO) enrichment analyses were performed using the DAVID online tool to annotate biological process, cellular component, and molecular function of DEGs. Additional information on the DEGs was obtained from IPA, cBioPortal, and Gene Expression Profiling Interactive Analysis (GEPIA). Protein-protein interaction (PPI) networks of the DEGs were constructed using Search Tool for the Retrieval of Interacting Genes (STRING) (16–19).
Immunohistochemistry (IHC)
Formalin fixed paraffin embedded (FFPE)-tissues of VSCC, HSIL, dVIN, and normal vulva were retrieved from the archives of Department of Pathology, Erasmus MC. Histology of all tissues was reviewed by two pathologists (SDG and PCEG). Patient data were anonymized and patient materials were handled following the guidelines of World Medical Association Declaration of Helsinki.
For performing IHC, DEGs were selected-i) that were expressed in the cytoplasm or nucleus and ii) for which primary antibodies were commercially available. In addition, for all samples, IHC was performed with p16 to determine the HPV-status, and with p53 to confirm the histological diagnoses. Sequential sections of 4 µm-thickness were prepared from the FFPE-tissues and automated IHC was performed using the Ventana Benchmark ULTRA (Ventana Medical Systems Inc.), following the manufacturer's instructions (Data S1 and Table SI).
The IHC markers were scored as follows: For the IHC markers of DEGs, the percentage of cells showing staining, irrespective of the intensity of staining, was assessed manually. In addition, the intensity of staining (weak, moderate, and strong) and the distribution of staining within the epithelium was recorded. p16-expression patterns were scored as block-type or non-block-type (patchy), following the guidelines of Lower Anogenital Squamous Terminology Standardization Project (LAST) (16). Block-type p16-expression, i.e. diffuse, continuous, moderate-to-intense nuclear and/or cytoplasmic staining in ≥1/3rd of the epithelial thickness is considered to be a reliable surrogate marker of high-risk HPV-infection (20). p53-expression patterns were scored as p53-mutant or p53-wild-type following descriptions in recent literature (10,21). p53-mutant patterns have been reported to accurately reflect the presence of TP53 mutations (10). p53-mutant patterns include basal to para-basal/diffuse overexpression, basal overexpression, or aberrant negative/null-pattern. p53-wild-type patterns include scattered heterogeneous basal and/or para-basal expression, and scattered mid-epithelial expression with basal sparing. The latter p53-wild-type pattern is associated with HPV-related lesions (10,22).
Ethics statement
This study was conducted in accordance with the guidelines of the Dutch Federation of Biomedical Scientific Societies (www.federa.org/codes-conduct), which state that no separate ethical approval is required for the use of anonymized residual tissue procured during regular treatment.
Results
Dataset analyses
From GSE38228, 3 samples were identified as HPV-related VSCC and 3 samples were identified as HPV-independent VSCC. A total of 342 genes (244 upregulated and 98 downregulated) were found to be differentially expressed with statistical significance only in HPV-related VSCC. A total of 382 genes (203 upregulated and 179 downregulated) were found to be differentially expressed with statistical significance only in HPV-independent VSCC. From the 2nd dataset, 3 samples were identified as HPV-related VSCC and 2 samples as HPV-independent VSCC. A total of 7005 genes were differentially expressed with statistical significance in HPV-related VSCC, and 4,283 genes were differentially expressed with statistical significance in HPV-independent VSCC.
Combining both datasets, for HPV-related VSCC, 88 DEGs were identified that were similarly regulated with statistical significance. This comprised 69 upregulated and 19 downregulated DEGs; signal transducer and activator of transcription 1 (STAT1) was one of the upregulated DEGs. For HPV-independent VSCC, 46 DEGs were identified that were similarly regulated with statistical significance. This comprised 16 upregulated and 30 downregulated DEGs; nuclear factor IB (NFIB) was one of the downregulated DEGs. The PPI networks of these DEGs are visualized in Figs. 1 and 2, and the DEGs along with their subcellular locations, functions, and related canonical pathways are listed in Tables SII and SIII.
The DEGs identified for HPV-related VSCC mainly participate in response to stimulus and regulation of cellular and biological processes (Table I). As for the molecular function, these DEGs are mainly involved in binding with ions or signaling receptors (Table II). The cellular component of these DEGs include cytoplasm and extracellular region.
Table I.GO enrichment analysis of the differentially expressed genes for human papilloma virus-related vulvar squamous cell carcinoma. |
Table II.Functional annotation analysis of the differentially expressed genes for human papilloma virus-related vulvar squamous cell carcinoma. |
The DEGs identified for HPV-independent VSCC mainly participate in regulation of cellular and metabolic processes (Table III). As for the molecular function, these DEGs are mainly involved in protein and ion binding (Table IV). The cellular component of these DEGs include membrane-bound organelles and the cytoplasm.
Table III.GO enrichment analysis of differentially expressed genes for human papilloma virus-independent vulvar squamous cell carcinoma. |
Table IV.Functional annotation analysis of differentially expressed genes for human papilloma virus-independent vulvar squamous cell carcinoma. |
Immunohistochemistry
Primary antibodies for performing IHC were commercially available for i) STAT1, one of the upregulated DEGs, and ii) NFIB, one of the downregulated DEGs. IHC was performed on 11 VSCCs, 6 dVINs, 6 HSILs, and 7 normal vulva tissues; these were from women with a median age of 72.5 years (range, 26–90 years). Immunohistochemical expression of p53, p16, NFIB, and STAT1 are presented in Tables V and VI. For NFIB and STAT1, the IHC patterns observed in the tissues are described below, and the distribution of expression is depicted in Fig. S1.
STAT1
Normal vulvar tissue (n=7): Five showed diffuse, cytoplasmic STAT1-expression of moderate-to-strong intensity, across full epithelial thickness; two showed focal STAT-1 expression of moderate-to-strong intensity.
dVIN (n=6), HSIL (n=6), HPV-related VSCC (n=5), HPV-independent VSCC (n=6): All showed diffuse, cytoplasmic STAT1-expression of moderate-to-strong intensity, across full epithelial thickness.
NFIB
Normal vulvar tissue (n=7): All showed strong, diffuse, nuclear NFIB-expression, predominantly along the basal layers, which occasionally extended to the para-basal layers.
HSIL (n=6): All showed strong nuclear NFIB-expression along the basal layers and occasionally in the para-basal layers. Staining in the basal layer was discontinuous, and expression in the para-basal layers was primarily seen only at the tips of rete ridges.
HPV-related VSCC (n=5): Two were completely negative, and 2 were predominantly negative, showing only focal, weak, nuclear NFIB-expression along the periphery of the tumor cell nests. One VSCC showed NFIB-expression of moderate intensity along the periphery of the tumor cell nests.
dVIN (n=6): One dVIN was completely negative and 5 showed only focal, weak, nuclear NFIB-expression.
HPV-independent VSCC (n=6): One was completely negative, and 5 were predominantly negative, showing only focal, weak, nuclear NFIB-expression along the periphery of the tumor cell nests.
Immunohistochemical expressions of p53, p16, STAT1, and NFIB in normal vulvar tissue, HSIL, dVIN, and VSCC (both subtypes) are demonstrated in Figs. 3–7.
Discussion
In this study, we utilized bioinformatics tools to gain insight into VSCC carcinogenesis, and to identify potential biomarkers that may have diagnostic, prognostic, or therapeutic applications. For both subtypes of VSCC (i.e., HPV-related and HPV-independent) we identified a set of DEGs that appeared to be similarly regulated (up or down) in two independent gene expression microarray datasets. We found that the majority of DEGs that were identified for HPV-related VSCC are involved in the immune response, whereas those identified for HPV-independent VSCC were involved in second messenger signaling-this provides support for the dual pathogenesis of VSCC.
We studied the expression of two of the DEGs, i.e. NFIB and STAT1, that were found to be similarly regulated in both datasets, in whole tissue sections of VSCCs, dVINs, HSILs, and normal vulvar tissues, by performing IHC. NFIB was identified to be downregulated in HPV-independent VSCC, and STAT1 was identified to be upregulated in HPV-related VSCC. Neither of these markers has been previously studied for VSCC or its precursor lesions.
NFIB showed strong, nuclear expression in the basal and para-basal epithelial layers in normal vulvar tissue, whereas, in dVIN and both subtypes of VSCC, NFIB was either completely negative or minimally expressed. NFIB expression was also reduced in HSIL in comparison with normal vulvar tissue, but to a lesser extent than that in dVIN and VSCC.
NFIB is a transcription factor which has tumor suppressive, as well as, oncogenic potential (23). In cervical SCC and head-and-neck SCC (HNSCC), NFIB-expression has been observed to be lower than in normal tissues from the corresponding sites (23). Furthermore, lower levels of NFIB-expression have been reported to correlate significantly with worse prognosis for both of these malignancies (23). Interestingly, NFIB is a key regulator of the aryl hydrocarbon pathway, which we previously identified to be involved in HPV-independent VSCC (2). In addition, high-confidence proximity interactions have been reported between NFIB and SOX2 (24); SOX2 is a cancer-stemness related transcription factor that is overexpressed in dVIN and VSCC (25). In view of these observations, we believe that the role of NFIB in VSCC and its potential as a therapeutic target deserve further investigation. In addition to SCCs, genomic alterations of NFIB have been detected in several other malignancies, as shown in Fig. S2.
Unlike NFIB, no discernable difference was observed in immunohistochemical expression of STAT1 between normal vulvar tissue, dVIN, HSIL, or VSCC (both subtypes). For all tissue types, diffuse, cytoplasmic STAT1-expression of moderate-to-strong intensity was noted across full epithelial thickness. STAT1 is a component of the Janus kinase (JAK)-STAT signaling pathway, and can act as an antimicrobial mediator, a tumor suppressor, or a promotor of tumor progression (26). Aberrant expression of STAT1 in HPV-related lesions is considered to reflect activation of the JAK-STAT pathway as a consequence of the inflammatory response induced by HPV (26).
Our results regarding IHC-expression of STAT1 were in contrast to those of a recent study, which reported a higher STAT1-expression in cervical intraepithelial neoplasia (CIN) than in normal cervical epithelium, and deduced an association of increased STAT1-expression with malignant progression of CIN (26). Since STAT-1 expression is regulated by a complex network of interferons, we speculate that the difference in expression between vulvar and cervical tissue could be ascribed to the dissimilar microenvironments of these anatomical sites. Similarly to NFIB, genomic alterations of STAT1 have been detected in several malignancies, as shown in Fig. S3.
This study was an attempt to leverage bioinformatics to identify DEGs in VSCC. We identified NFIB as a downregulated gene in VSCC, and observed that its immunohistochemical expression was reduced in both subtypes of VSCC. Hence, we believe that the relevance of NFIB as a diagnostic/prognostic biomarker deserves further exploration. However, an apparent limitation of this study is that the DEGs were identified from datasets consisting of small sample sizes, and IHC was also performed on a limited set of tissues. Further experiments are needed to confirm the function of these DEGs in VSCC and to validate their immunohistochemical expression in vulvar tissues.
Nevertheless, we hope that our results will instigate further research into VSCC carcinogenesis and pave the path for unravelling novel biomarkers of VSCC and its precursor lesions.
Supplementary Material
Supporting Data
Acknowledgements
The authors would like to thank Dr Leen J. Blok (Erasmus MC) for the use of data on gene expression analysis.
Funding
No funding was received.
Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
Authors' contributions
SD, PCEG, TPPVDB, SMAS, LAMS, PJVDS, SK and FJVK conceived and designed the methodology of the current study. SMAS, PJVDS analyzed the data. SD, SMAS, PJVDS and FJVK confirm the authenticity of all the raw data. TPPVDB and LAMS performed the experiments. SD, PCEG, TPPVDB, SS, LAMS, HCVD and PJVDS curated the pathological and clinical data. SD drafted the manuscript, constructed the graphs and arranged the histology figures. PCEG, SK and FJVK supervised the current study. All authors reviewed and edited the manuscript and read and approved the final manuscript.
Ethics approval and consent to participate
The current study was conducted in accordance with the guidelines of the Dutch Federation of Biomedical Scientific Societies (www.federa.org/codes-conduct), which state that no separate ethical approval is required for the use of anonymized residual tissue procured during regular treatment. This is also part of the standard treatment agreement at Erasmus MC.
Patient consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
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