Introduction
Cervical cancer is the most common gynecological malignancy and ranks second in cancer-related mortality among women in India. In addition, according to GLOBOCAN 2022 estimates, cervical cancer is the fourth most common cancer among women worldwide. The estimated age-standardized incidence and mortality rates of cervical cancer per 100,000 females are 662,301 (6.9%) and 348,874 (8.1%), respectively (1). Major contributing factors include persistent infection with high-risk (HR) human papillomavirus (HPV), early onset of sexual activity, long-term use of oral contraceptives (>5 years), multiple sexual partners and lifestyle factors including consumption of alcohol and tobacco, ultraviolet light exposure, poor nutrition and a sedentary lifestyle (2).
HPV plays a major role in the pathogenesis of cervical cancer. There are >200 different subtypes of HPV, of which 40 strains are known to be sexually transmitted and associated with cervical, vulvar, vaginal and anal diseases (3). However, persistent infection with HR HPV subtypes, particularly HPV-16, -18, -31, -33, -35, -45, -52 and -58, leads to the dysregulated proliferation of healthy cervical cells, resulting in pre-malignant lesions that may become malignant over time (4). Among these, HPV-16 and -18 account for >70% of all cervical cancer cases (5).
Population-based studies have shown that the incidence of new cases of cervical cancer has declined over the past 20 years, primarily due to increased cervical cancer screening and vaccination (6,7). Despite World Health Organization recommendations, only 100 countries have included HPV vaccination in their national immunization programs (8). The implementation of HPV vaccination has led to significant reductions in HPV infection rates, anogenital wart diagnoses and high-grade cervical lesions among women, and reported to provide herd immunity benefits in boys and older women (9). However, in a number of low- and middle-income countries, including India, the integration of HPV vaccination into national immunization programs has not yet occurred (10,11). A major issue is the affordability of the currently available HPV vaccines.
The HPV genome is approximately eight kilobases in length and consists of double-stranded DNA. It encodes six early genes (E1, E2, E4, E5, E6 and E7) involved in viral replication and oncogenesis, along with two late genes (L1 and L2) contributing to capsid formation. The early genes regulate HPV genome replication, transcription, cell cycle progression, cell signaling and structural modifications in infected cells, while late genes are required for virus propagation (12). A number of studies have demonstrated the impact of E1, E2, E4, E6 and E7 oncoproteins on cervical cancer (13-16). HPV 16 E5 is a hydrophobic protein comprising 83 amino acids that localizes in the cell membrane, endosomes and Golgi apparatus (17). It impairs the degradation of activated epidermal growth factor receptor (EGFR) by interfering with the acidification of late endosomes (18-20). HPV16 E5 downregulates the expression of the tumor suppressor proteins p21 and p27, both of which are cyclin-dependent kinase (CDK) inhibitors that regulate the cell cycle by inhibiting CDK activity.
In vitro studies have demonstrated that HPV16 E5 increases the malignancy of cervical cancer cells by promoting proliferation, migration and actin cytoskeleton-mediated invasion (21). One such study showed that HPV16 infection leads to an increase in the expression levels of the Met receptor, a crucial growth factor receptor involved in tumor cell motility, invasion and metastasis. This upregulation of Met is primarily mediated by the viral oncogene E5, with a lesser contribution from E6. The mechanism by which E5 induces Met expression involves the EGFR. E5 increases EGFR expression at the mRNA level, and this upregulation is implicated in the induction of Met (22). This suggests a complex crosstalk between the EGFR and Met signaling pathways mediated by HPV oncogenes (23). Furthermore, another study proposed that the known antiviral agent rimantadine could be utilized to counteract this resistance mechanism, thereby improving treatment outcomes for patients with head and neck cancer. The study suggested that rimantadine may have the potential to enhance the efficacy of immunotherapy by inhibiting the action of HPV E5 and restoring the sensitivity of tumors to immune checkpoint blockade (24).
Pertinently, novel approaches for improved diagnostics, effective vaccines and increased awareness among women from lower socioeconomic backgrounds are required for effective cervical cancer management. The present study performed an in silico analysis focused on the E5 oncoprotein of HPV and explored the hub genes and related signaling pathways that contribute to the progression of cervical cancer. The expression of certain hub genes was validated using reverse transcription-quantitative PCR (RT-qPCR). The findings from this study may provide insights into the molecular etiopathogenesis of cervical cancer, which could be useful in the development of novel, high-efficacy diagnostics and vaccines.
Discussion
Oncoproteins are central to HR-HPV pathogenesis. Among the six early HPV genes, E6 and E7 have been well characterized, while the roles of other early genes, particularly E5, remain underexplored. E6 and E7 have been shown to disrupt cell cycle regulation in infected cells by targeting retinoblastoma protein and p53(46), thereby blocking p53-dependent cell-cycle checkpoints and promoting unregulated cancerous growth (47). In vivo studies suggest that HPV E5 supports the oncogenic activities of E6 and E7, thereby contributing to malignant progression (48,49). E5 is active during the early stages of viral infection (50). Further HPV E5 has been observed to activate mitogen-activated protein kinase (MAPK), p38 and ERK1/2 in human keratinocytes in an EGF-independent manner (51). This MAPK activation upregulates the transcription of c-fos and c-jun, which in turn stimulates the transcription of E6 and E7 (52-54).
Given the relevance of HPV E5, the bioinformatics analysis in the present study was focused on its role in cervical cancer. A comprehensive bioinformatics analysis of the microarray datasets GSE26888, GSE26511, GSE9750, GSE5787 and GSE7803 was performed, which led to the identification of 8,202 DEGs. Functional enrichment analysis showed that these DEGs are associated with 19 MFs, 40 BPs and 22 CCs. Out of the 19 MFs, top five were ‘kinase’, ‘activator’, transferase’, ‘tyrosine-protein kinase’ and ‘RNA-binding’. Major BPs included ‘host-virus interaction’, ‘protein transport’, ‘biological rhythms’, ‘apoptosis’ and ‘innate immunity’, while major CCs included ‘cytoplasm’, ‘nucleus’, ‘Golgi apparatus’, ‘mitochondrion’ and ‘cytoskeleton’. In addition, KEGG pathway analysis revealed that the DEGs were involved in 175 pathways, with the most notable being the ‘MAPK signaling pathway,’ ‘PI3K-Akt signaling pathway’ and ‘pathways in cancer.’ Finally, 10 hub genes, namely NUP160, NUP85, NUP205, SEC13, NUP50, NUP37, NUP98, NUP88, NUPL1 and NUP133, were found to be significantly associated with HPV E5 oncoprotein in cervical cancer pathogenesis. Notably, these hub genes are also implicated in important biological pathways such as ‘transport of the SLBP independent mature mRNA’, ‘cell cycle, mitotic’, ‘EML4 and NUDC in mitotic spindle formation’ and ‘gene silencing by RNA’.
The roles of some of these hub genes in different cancers have been reported previously. For example, NUP205 has been shown to increase nuclear c-Myc levels, thereby promoting the proliferation of lung cancer cells (55). In papillary thyroid cancer, HOTAIR-mediated metastatic progression has been observed to be regulated by the miR-488-5p/NUP205 axis (56). Furthermore, in colorectal cancer, the expression of NUP205 in tissues adjacent to the cancer has been suggested to be a predictor of relapse risk (57). The roles of NUP50 and NUP153 in breast cancer and bladder cancer have also been reported in previous studies (58,59). Elevated NUP37 expression has been associated with a low survival rate in patients with gastric cancer (59). NUP88 has been shown to inhibit the Notch signaling pathway in lymphatic leukemia by blocking the nuclear export of the recombination signal-binding component of this pathway (60). Furthermore, the upregulation of NUP88 overexpression has been observed in cases of highly aggressive breast cancers and cervical cancer cases (61,62). Lastly, NUP85 expression has been reported to be upregulated in pancreatic tumors (63).
Analysis of the hub genes using cBioPortal revealed significant co-occurrence for three sets of genes, namely: NUP50 and NUP98; NUP160 and NUP50; and NUP160 and NUP98, suggesting that variations in these genes may not occur in the same samples but might be present in different cancer cohorts, indicating their involvement in multiple pathways for cancer initiation and development (64,65). The observed co-occurrence supports the hypothesis that these genes do not act independently, and their protein products are likely to be interdependent and involved in shared biological pathways for the execution of specific cellular and molecular functions. To validate this finding and to investigate the relationship between these genes in cervical cancer, the expression levels of the NUP50, NUP98, and NUP160 genes in three cervical cancer lines were determined by RT-qPCR. The results revealed that all three genes were significantly upregulated in the HPV-positive cell lines HeLa and SiHa compared with the HPV-negative cell line C33A. NUP50 is a nucleoporin notably localized in the nuclear pore complex and the nucleoplasm. The deletion of NUP50 has been shown to cause abnormalities in the expression of p27Kip1, a CDK inhibitor protein that regulates cell cycle progression and cell proliferation, particularly during development of the neuroepithelium (65). The upregulation of NUP50 observed in HeLa and SiHa cells may contribute to the dysregulated growth of these cells through the dysregulation of this pathway via interaction with the HPV E5 protein.
NUP98 plays a regulatory role in gene expression, particularly for genes associated with development regulation and the cell cycle. In addition to being a component of the nuclear pore complex, its presence or absence in the nucleoplasm appears to modulate transcriptional activity (66). The dynamics of NUP50, NUP98 and NUP160 show HPV strain-specific expression, which warrants validation in further functional studies.
In addition, an OncoPrint analysis performed using cBioPortal was conducted to observe the type of mutations, including deep deletions, amplifications and missense mutations, and mRNA expression changes of these hub genes in cervical cancer. Among these, NUP133 showed the highest frequency of genetic alterations (26%), followed by NUP98 (14%) and NUP85 (13%). However, while overall survival and progression-free survival analyses of the combined top 10 hub genes did not show a significant association, survival analyses of individual hub genes demonstrated that NUP85, NUP88 and NUPL1/NUP58 were significantly associated with an improved prognosis in cervical cancer.
Cross-validation was performed using the bioinformatics tools HPA and GeneMANIA. The expression of the identified key genes was examined in eight cervical cancer cell lines using HPA data, which revealed that their expression was altered in the presence of HPV; however, patient IHC data was incomplete, with the exception of that for NUP50. The GeneMANIA analysis was performed to highlight physical interactions, co-expression, co-localization, genetic interactions, and pathway associations, and indicated that the key NUP genes influence the immune system (67).
The in silico and RT-qPCR expression analyses performed in the present study suggest that the HPV E5 oncoprotein may play a role in cervical carcinogenesis by modulating the expression of the identified NUP genes. However, this requires validation using functional assays to elucidate the precise role of HPV E5 in the molecular etiopathogenesis of cervical cancer.
To the best of our knowledge, this is the first study to implicate NUPs as being significantly involved in HPV E5-mediated cervical cancer progression. NUPs are essential components of the nuclear pore complex and regulate the flow of various molecules across the nuclear envelope by interacting with importin and exportin proteins (59). The findings of the present study may be helpful in the development of potential biomarkers for the early diagnosis of cervical cancer and the identification of novel vaccine targets. However, the publicly available datasets used in this study may contain inherent experimental variations that could not be accounted for. Despite this limitation, the data generated and prior evidence suggest that the role of the E5 oncogene in cervical cancer is noteworthy and merits further exploration.
While the current study provides valuable insights through the integrative analysis of five transcriptomic datasets, it is important to acknowledge certain limitations. Firstly, the analysis provide a collective interpretation of all datasets to maintain consistency with the overarching study objectives. As a result, individual dataset-level differences and pathway enrichment analyses specific to dataset pairs were not performed. Although this integrative approach was advantageous for identifying broader trends, it limited the ability to uncover dataset-specific biological variations and enriched pathways that may offer additional mechanistic insights. Secondly, the current study used the GEO2R tool to perform the differential gene expression analysis. While efficient for pre-processed datasets, this tool does not offer the statistical robustness of DESeq2, which provides superior normalization, improved handling of low-expression genes, and accounts more effectively for batch effects; however, its application requires a specific raw data file format, which was not available for the current study. The inability to use DESeq2 may have limited the precision of the identification of DEGs and hub genes, particularly in complex RNA-seq datasets. Future studies may benefit from the use of DESeq2 or similar tools to enhance analytical depth, particularly where raw count data is available. Finally, to validate the results of this study, detailed in vitro, in vivo and ex vivo will be essential in the future. Addressing the limitations of the present study through dataset-specific enrichment analysis, more advanced statistical methods and comprehensive preclinical research represents an important direction for future research.
Although various studies have shown HPV E5 oncoprotein to be responsible for initial virus propagation, there is a paucity of evidence regarding its exact role in cervical cancer pathogenesis. The present study highlights the importance of HPV E5 in the development of cervical cancer. Publicly available online databases were searched and an integrated bioinformatics analysis was performed, which revealed 10 hub genes, of which three, namely NUP50, NUP98 and NUP160, were shown to be significantly associated with an improved prognosis in cervical cancer. These findings highlight the role of nucleoporins in cervical cancer development; however, they require functional validation, to gain a more comprehensive understanding of the molecular mechanism and potentially aid in the development of novel diagnostics and vaccines.