Here we have studied the expression of selected proteins implicated in cell adhesion, motility, and differentiation in cervical dysplasia associated with high-risk human papillomavirus infection. We have previously reported large-scale gene and microRNA profiling in epithelial cells expressing the HPV 16 E5 oncogene [10, 11]. In those studies the E5 protein was found to affect crucial cellular pathways including cell adhesion, cell motility, extracellular matrix and mitogenic signaling, and those effects were partially shown to be mediated by microRNA regulation [10, 11].
In the present work we sought out to study whether altered cellular gene expression profiles found in an experimental setting would apply to human cervical disease. We studied altogether 50 samples representing human cervical disease of different grades, together with four samples representing normal squamous epithelium and two from columnar cervical epithelium. The aim of the study was to perform preliminary biological validation of gene expression microarray findings in human disease, and to search for putative feasible biomarkers for disease diagnostics. The study was limited by the availability of reliable antibodies to cellular proteins for immunohistochemical staining. Further, the sample number per histological grade is rather small and thus our results are rather of suggestive nature. However, we were able, by using tissue samples from human cervical disease, to validate trends of altered expression of several genes which were first identified in microarray.
Matrix metalloproteinases are proteins that degrade the extracellular matrix and are thus essential in a number of physiological processes and pathological conditions including embryonic development, differentiation, tissue remodeling and cancer , also in cervical cancer . Specifically the involvement of MMP-2, MMP-9 and MMP-14 in cervical carcinogenesis has been reported [17–19]. Expression of MMP-7 has been found to correlate with invasive property and advanced tumor stage of head and neck squamous cell carcinoma , and endometrial  and gastric adenocarcinoma . This seems to be tumor type specific, however, since most cervical neoplasia and carcinoma cases were found negative for MMP-7 . Interestingly, in a previous study no expression of MMP-12 or MMP-16 was found in HPV harboring cervical carcinoma cell lines .
In the present study we found increasing staining for MMP-16 along with the development of cervical dysplasia, in agreement with a number of previous studies, whereas MMP-7 expression remained virtually similar. Although the individual role of the E5 oncogene in the carcinogenic process is not fully understood, this seems to disagree with our previous finding of reduced MMP-7 and MMP-16 mRNA expression in monolayer cells expressing the E5 protein , albeit protein expression in that study remained unchanged, similar to what we have shown here for MMP-7 in tissue samples. That result may have been due to increased half-life of the transcripts or increased degradation of the protein. Altogether, effects of the E5 oncogene as seen in microarray may only represent early and/or partial consequences of human papillomavirus in infected epithelial cells. In this work, both MMP-7 and MMP-16 were found to be expressed in the nuclei of basal and suprabasal cells of normal squamous epithelium and condyloma. Most reports describe MMP localization at the level of tissue, not within cells, and only a few papers discuss the function of nuclear MMP localization. However, nuclear localization of proteolytically active MMP-2 in cardiac myocytes, and MMP-3 in cultured cells and in human liver tissue has been shown [24, 25]. MMP-2 cleaves PARP in the nucleus of cardiac myocytes, and the authors speculate that this might inhibit DNA damage repair by PARP . The effects of PARP inhibition in cancer, however, have been shown to be contradictory . Nuclear MMP-3 seems to be involved in the induction of apoptosis . Although our findings do not allow further speculation due to modest changes in MMP expression, the putative functions of nuclear MMP’s are worth further studies considering the process of cervical carcinogenesis.
Keratin-8 is often paired with keratin-18 and is expressed in simple epithelia . Here we show reasonably strong scattered expression of cytokeratin-8/18 in severe intraepithelial neoplasia. The expression of CK8/18 in squamous epithelium is an unresolved issue, although its expression has been previously shown in high-grade CIN and squamous cell cervical cancer [27–29]. Poor prognosis has been correlated with cytokeratin 8/18 expression in squamous cell carcinoma of the oral cavity . CK8/18 expression was shown in carcinoma induced by inoculating nude mice with tumorigenic HPV 16-immortalized keratinocytes . Our results agree with the previous data, because normal squamous epithelium, low-grade lesions, and even CIN2 were basically negative for CK8/18, whereas strong although scattered expression was shown in CIN3.
Laminin-5 enhances cellular migration and tumorigenicity, and it has been shown to be overexpressed in cervical cancer . High-risk HPV E6 oncogene has been previously shown to upregulate laminin-5 receptor expression in cervical cancer cells . This takes place by E6-mediated downregulation of human miR-218, leading to upregulation of its target gene LAMB3, which is a component of the laminin-5 receptor expressed in the basal lamina . On the contrary, in our study the staining for laminin was strongest in CIN1, whereas in other tissues laminin was seen only in the basal lamina. Unfortunately exact information about the reactivity of the antibody was unavailable and thus we cannot further speculate about the role of laminin in CIN1. This is, however, an extremely interesting finding, considering the possible early events towards carcinogenesis being evident already in a low-grade lesion.
Carcinogenesis essentially involves downregulation of E-cadherin and disruption of E-cadherin–beta-catenin complexes in adherens junctions . Along with the malignant development, the E-cadherin–beta-catenin complex at the plasma membrane is degraded and beta-catenin is transported to the nucleus where it acts as a transcription factor in the Wnt signaling pathway and contributes to malignant development. Differences in intercellular junctions even among morphologically similar cells may be a manifestation of a crucial event in cervical carcinogenesis [34, 35]. The expression of E-cadherin and beta-catenin in HPV-associated cervical neoplasia is, however, equivocal. Decreased and cytoplasmic expression of those proteins in cervical cancer has been reported [36–39]. Leong et al. showed weakening and dysregulated E-cadherin expression in HPV harboring lesions . Those authors showed that although downregulation of E-cadherin has previously been associated with the development of neoplasia and cancer, it seems to have little to do with the carcinogenic process and seems to be unrelated to the ability of the E6 and E7 proteins to bind and degrade pRb or p53 . Quite the opposite, in the present work we found increased membrane and cytoplasmic staining for both E-cadherin and beta-catenin in high-grade cervical lesions. In agreement with this, Samir et al. reported increased E-cadherin expression with increasing CIN grade .
We have previously shown increased expression of E-cadherin and beta-catenin due to E5 expression in epithelial cells . We further observed downregulation of cellular miR-324-5p, which we showed to putatively target both E-cadherin and beta-catenin, and thus its downregulation would lead to increased expression of the target proteins . In the present work, in agreement with previous observations, we observed increased expression of E-cadherin and beta-catenin due to E5, E6 and E7 oncogenes in three-dimensional collagen raft cultures. This is in agreement with the increased expression found in high-grade human cervical tissue. Interestingly, most intense staining in E5-expressing raft cultures was seen in the most differentiated layers of the raft epithelium, contrary to normal tissue, condyloma or CIN1, where most intense staining was seen in the bottom layers of the epithelium. In raft cultures expressing the E6 and E7 oncogenes the strongest staining was seen in the lower part of the raft epithelium, similar to human tissue. This repeated observation might be explained by decreased expression of adhesion molecules towards fully differentiated and desquamating cells which are found in normally maturing but not in neoplastic epithelium.
Extracellular matrix-degrading proteins may be involved in modification of intercellular junctions. It has been speculated that MMP-7 expressed on the surface of carcinoma cells may cleave E-cadherin and facilitate the detachment of carcinoma cells from the site of primary tumor , although we did not see this inverse relationship of expression in our work. The expression of the secreted extracellular matrix-degrading proteinase MMP-7 has been shown to be upregulated by beta-catenin in colon tumor cell lines, and a crucial role prior to the onset of the invasive phenotype has been suggested . We observed increased membrane and cytoplasmic staining for beta-catenin along with the severity of the lesions, including AIS, whereas MMP-7 expression remained virtually similar. Regulation of various cellular functions participating in carcinogenesis is complex. For example, the role of integration of the HPV genome into the chromosome in the differential expression of cellular proteins is poorly understood.
For normal epithelial development, basal epithelial cells have to detach from integrin and laminins of the basement membrane, cease to proliferate, and enter the differentiation program. In epithelial dysplasia or cancer, cells do not differentiate but continue to proliferate. Many of the phenomena seen in cervical intraepithelial neoplasia and cancer are the results of viral oncoprotein functions. A number of observations based on cervical morphology and tissue staining have led to better understanding of cervical pathogenesis. Also, careful examination of viral functions in experimental systems contribute to our knowledge of carcinogenesis and, importantly, may provide novel biomarkers for the diagnosis, follow-up and cure of cervical disease.