We performed a comprehensive analysis that stems from data obtained by proteomic techniques, using perspectives of biological networks and the annotated data of the human genome and large-scale sequencing techniques available to the public as a tool for building models to infer biological behaviors (Figure ).
As defined in this study, the "central core of cervical cancer" does not describe the immortalization process directly; rather, it represents processes involved in maintaining a viable and proliferating cell. Table presents the biological processes that are enriched in our core set of proteins and as can be seen, these are suggestive of a malignant phenotype, where the main GOs are those related to UPR, metabolism, catabolic processes, maintenance of cell-location and cell death. These observations are consistent with our initial conjectures, since we considered that using the HaCaT cell line as a control would allow us to distinguish between immortalization and transformation, because HaCaT is an immortalized cell line with a stable kariotype and phenotype, which is non-tumorigenic.
One of the studies that uses the HaCaT cell line as a control was performed by Choi, et al
]. The main difference between the latter study and our work, is that they used cervix biopsies; this implies that there are important differences in the intracellular and extracellular contexts of the biological model. Likewise, even though the methodology they utilized only considers samples with the same pathologic diagnosis, it is likely that there is still an important amount of heterogeneity in their samples due to inter-individual differences. As expected, we only found one common protein among the results of both studies: Vimentin. However, they were able to identify a number of proteins that strengthen our findings such as Amy-1, which stimulates c-Myc's E-box dependent transactivation activity [43
]; and Miz-1, which must be inhibited in order for c-Myc to mediate apoptosis [44
]. Another result that supports our findings is that they observed a downregulation of 14-3-3σ, which plays an opposite role in cell growth, compared to 14-3-3ζ [45
]. Finally, they also found a clear overexpression of chaperone proteins, different from the ones in our central core.
The acquisition of the reported interactions between the identified proteins provided a first insight of the key elements in the neoplastic dynamics, and became the meter that allowed us to judge the validity of later analysis, by seeking an overall agreement between the original and the extended network. It is noteworthy that for the small network the highest interconnected node was 14-3-3ζ, and this was not lost when we expanded the network. Also, both networks are enriched in similar processes.
When analyzing the extended network it is apparent that 6 of the members of the 14-3-3 protein family (ζ, γ, β, τ, σ and ε) feature among the highest interconnected nodes. This is consistent with the biological role of the members of this protein family as signal transduction molecules [46
]. The identification of 14-3-3ζ and its role as a hub and as a bottleneck in the PPI network is particularly relevant because it is known to be involved in three important cellular processes: cell cycle regulation, signal transduction and regulation of apoptosis [47
Furthermore, we noticed that the 14-3-3ζ protein, which features as the highest interconnected node of both our networks, did not posses any ChIP-Seq peak near the transcription start site of its encoding gene. Upon more careful inspection throughout the entire length of the gene, we noticed that there appears to be a regulatory region inside the transcript. Importantly, we found binding sites of c-Myc, Max, E2F1 and Pol2 very close to each other. This has lead us to believe that one of the mechanisms that lead to 14-3-3ζ's overexpression is mediated by c-Myc and E2F1 (Figure ).
Figure 5 Model of Action of c-Myc, E2F1 and 14-3-3ζ. Model of the downstream events product of the overexpression and/or amplification of c-Myc, and its collaboration with the transcription factor E2F1. c-Myc promotes the expression of proteins that lead (more ...)
It was previously reported by Jonsson and Bates [13
] that cancer proteins exhibit a significantly different network topology when compared to proteins unrelated to cancer; as well as a higher ratio of promiscuous structural domains. This is in agreement with what is known about the members of the 14-3-3 protein family, which have been described as signal integrators, amplifying strong signals and filtering out weaker conflicting ones to achieve a meaningful, coordinate biological output, such as cell death or survival [51
Also, there is strong evidence that the overexpression of 14-3-3ζ promotes p53 degradation by the proteosomal route [52
], which is increased by the action of E6 in HPV-infected cells [7
]. An enhanced turn-over rate of the p53 protein and the pleiotropic effects consequence of the increased number of 14-3-3ζ proteins, which are involved in a large number of cellular processes, could be the reason why not all infections develop into cancer and most of them are subclinical: c-Myc could be activating 14-3-3ζ in a differential manner. Therefore, we consider that we posses sufficient evidence to suggest the role of 14-3-3ζ in cervical cancer cell lines as the determining factor that can ultimately dictate the fate of a cell, regardless of it being infected with HPV; and cause it to undergo cell cycle deregulation onto malignant transformation (Figure ).
Likewise, when we performed a biological pathway enrichment, which we consider the best way to perform a functional analysis, we found consistent results. To do this, we used the ConsensusPathDB site to find overrepresented KEGG pathways in the proteins of our extended network. The enrichment analysis showed an overrepresentation of survival factors of apoptosis pathways and focal adhesion that is related to invasion and metastasis, two of the most important and well-documented hallmarks of cancer; as well as various established cancer pathways like small cell lung cancer, prostate cancer, endometrial cancer and pathways in cancer (Table ). This was obviously expected and helps to strengthen our analysis, indicating that the central core of cervical cancer, and its subsequent expansion proposed in the PPI interaction network, still retain those proteins that are closely related to the neoplastic process.
However, there are other pathways that do not present such a straightforward connection to cancer, such as the neurotrophin and T-cell receptor signaling pathways, bacterial infections and proteasome pathways. We can only speculate about their meaning, as the cellular stress, chronic inflammation, turnover of proteins, cell survival and evasion of the immune system are other features related to the progression and maintenance of tumorigenesis.
As a way to understand the underlying regulatory dynamics of the system, we also performed an enrichment analysis of transcription factor binding sites among the promoters of the genes of our extended network. We focused on the three transcription factors that complied with both of the following: those which were significantly overrepresented among the promoter regions of the genes of the proteins of our network and those in which the corresponding ChIP-Seq assays were carried out on the HeLa-S3 cell line http://genome.ucsc.edu/
. The resulting transcription factors are: E2F1, c-Myc and Max. We believe that together, these transcription factors bring an overall feeling of coherence to the network because the overexpression of c-Myc is widespread in human cancers, and specifically in cervical cancer [53
]. Moreover, there is evidence that E2F1 is activated by the E7 protein of HPV-16 in a pRB-independent manner [54
], apart from its pRB-dependent activation, which in the case of HPV infected cells is also promoted by pRB inactivation by viral oncoprotein E7. In the context of cervical cancer, it has been reported that an overexpression of E2F1 can drive quiescent cells through G1 into S-phase of the cell cycle, ultimately leading to apoptosis or neoplastic transformation.
E2F1 has been shown to be an inhibitor of c-Myc's activator, β-catenin, by means of the Wnt pathway [55
]. We also found a clear overrepresentation of the TCF4 transcription factor in the genes of our network. This contradicts our previous finding because TCF4 functions in the activation of the Wnt pathway, and therefore, of c-Myc. However, we had to consider that all the published works that point to E2F1 as an inhibitor of c-Myc have been performed in colorectal cancer. Likewise, the evidence of enrichment of TCF4 binding sites was performed in the HCT-116 cell line and so we do not posses information about the function of this transcription factor in cervical cancer.
It has been observed in human foreskin keratinocyte and fibroblast (HFK and HFF) cells that the genetic background of the cell type can be determinant for the outcome of altered gene expression. This is the case of HFF which cannot be immortalized by the mere addition of oncoproteins E6 and E7, unlike the HFK cells, due to a differential turnover of c-Myc [57
]. Moreover, as pointed by Bernards [58
] in the context of colorectal cancer pRB is more likely to be acting as an oncoprotein than as a tumor suppressor, which is clearly not the case in cervical cancer. Considering all the previous evidence, we can only infer that the proteins pRB and E2F1 are acting with opposing roles in cervical cancer, as compared to colorectal cancer.
On the other hand, there are reports of E2F1 and c-Myc overexpression in cervical cancer, which also correlate their expression with advanced states of the disease [59
]. Also, genetic studies of the E2F promoters have shown that these genes are induced by c-Myc, dependent on the E box sites. Coordinately, all data point to a model where c-Myc is activated by means different from the canonical pathways and is working with E2F1 to promote the neoplastic phenotype.
The possible scenario of molecular events in cervical cancer suggested by our study portrays the evasion of apoptosis mediated in two ways, first, the sequestration of the pro-apoptotic protein BAD by 14-3-3ζ [60
] and the overexpression of the anti-apoptotic protein Bcl-2 managed by the interaction of c-Myc and E2F1 [61
]. Evasion of apoptosis by these routes can be supported by the interaction of viral oncoproteins E6 and E7 with tumor suppressor proteins p53 and pRb, when there is persistent infection with high-risk HPV. Simultaneously, these oncoproteins are able to promote significant increase in proliferation, and immortalize cells through the activation of hTERT [62
The expression of E2F factors and cyclins facilitated by c-Myc, together E2F1 leads to a quick transition from the G1 to the S phase of the cell cycle, and the consequent boost in growth and cell proliferation [63
Finally, c-Myc overexpression allows a significant increase in cell proliferation by key roads, such as protein biosynthesis, central metabolism, the expression of transcription and cell cycle factors [64
] and expression of 14-3-3ζ. All of these contribute to facilitate the transition from G1 to S phase [65
]. c-Myc's mediated expression of Ezrin [66
] also promotes epithelial mesenchyma transition, facilitated by the overexpression of vimentin [67
] (Figure ).