Cervical cancer (CC) is an important health problem and is a leading cause of cancer mortality worldwide in women. [1] When exposed to and infected by one of the high-risk human papillomaviruses (HPV), vulnerable cervical epithelium may enter a complex multistep process and develop an invasive carcinoma. [2–4] The spectrum of histologic alterations during the intricate processes of multistep carcinogenesis can be classified as premalignant lesions, including low-grade and high-grade squamous intraepithelial lesions (SILs), and malignant invasive cervical cancers. [5] Despite its strong association with CC, HPV infection alone is not sufficient for the cervical epithelium to fully develop an invasive cervical cancer. Persistent HPV infection contributes to the development of SILs, with viral oncoproteins facilitating the dysregulation of cellular proliferation and the apoptotic process. However, additional accumulation of mutations, as well as epigenetic alterations in the crucial oncogenes and tumor suppressor genes, is required before these premalignant lesions fully transform into invasive cancers. [6]

The aim of this study was to evaluate DNA methylation status of cyclin A1 (CCNA1) in HPV-associated CC. CCNA1, a second A-type cyclin, has been shown to be essential for entry into metaphase of male meiosis I[7, 8] Consistent with this function, CCNA1 is highly expressed in testis and hematopoietic progenitor cells, but is present at low levels in most other tissues. [9] No phenotype other than male infertility has been reported in mice lacking CCNA1. [10] Surprisingly, several lines of evidence suggest that CCNA1 may be a potential epithelial tumor suppressor gene. First, the expression of CCNA1 has been demonstrated to be downregulated in several cancers, such as nasopharyngeal carcinoma and head and neck squamous-cell cancer (HNSCC). [11–13] Second, CCNA1 plays an important role in DNA double-strand break repair following radiation damage by activation of the non-homologous end-joining process that confers DNA stability. [14] Finally, the promoter, similar to several key tumor suppressor genes, is frequently hypermethylated in colon cancer and HNSCC. [13, 15]

Expression of CCNA1 has been shown to be correlated with the activation of TP53. In a HNSCC model, there is an inverse relationship between CCNA1 promoter methylation and TP53 mutation status in HNSCC tissues. [13] Similar to HNSCC, the majority of CC is of squamous cell origin and its molecular carcinogenesis strongly correlates with impaired TP53 function. [16–18] However, unlike HNSCC, the functional loss of TP53 in CC is not ascribed to gene mutation, but is processed by viral and host protein-protein interaction. CC is strongly associated with infection by high-risk HPV types and its oncoprotein E6 has the ability to associate with and neutralize the function of TP53. [17, 18] E6 binds to TP53 and catalyzes multi-ubiquitination and degradation of TP53. Consequently, the majority of CC cells have a wild-type TP53, but the protein levels are decreased. Therefore, in comparison with HNSCC, it was of interest to determine if CCNA1 is methylated in HPV-associated squamous cell CC.

The aim of this study was to determine if the CCNA1 promoter is methylated in CC and to elucidate how the epigenetic alteration occurs during multistep CC development. The experiments conducted comprised of: first, establishment of CCNA1 MSP; second, identification of the methylation status and correlation with expression in CC cell lines, normal cervix and CC; and finally, investigation of the frequency of methylation in normal tissues, high-risk HPV-associated low SILs, high SILs, microinvasive and invasive squamous cell CC.

CCNA1 methylation in CC cell lines

Duplex MSP for CCNA1 was designed according to the sequence in Figure 1A. The methylated sequence comprised of 46 bp and the non-methylated sequence, 67 bp, shown as the lower and the upper amplicons, respectively.

Previously, Carsten Müller-Tidow et al. [26, 27] extensively studied the role of CCNA1 methylation and found that CCNA1 was methylated in several non-expressing tumor cell lines, including HeLa. To confirm this particular finding in CC cell lines, we investigated methylation and expression in HeLa and SiHa cells. Our preliminary study in HeLa, HeLa(S), revealed complete non-methylation, which contradicts the previous report (Fig. 1B). To settle this controversy, we attempted to further evaluate additional CC cell lines, including HeLa(K) grown in a different laboratory, and SiHa. The result confirmed the Carsten Müller-Tidow et al. [26, 27] finding, in that the majority of Hela(K) cells, as well as all SiHa cells, were hypermethylated. CCNA1 RT-PCR confirmed the inverse relation between DNA methylation and gene expression. CCNA1 RNA levels were high, intermediate and low in HeLa(S), HeLa(K) and SiHa cells, respectively (Fig. 1). These data indicate that CCNA1 methylation is common in CC cell lines and its physiological role is to decrease gene expression. The absence of methylation in HeLa(S) might indicate a demethylation process that occurs under different cell culture and maintenance conditions.

We validated the reliability of this duplex MSP by performing calibration experiments using SiHa mixed with HeLa(S), CCNA1 completely hypermethylated and non-methylated cells, respectively (Fig. 2A). With at least three replicates for each experiment, the result demonstrates the consistency of the current approach, with minimal intra- and inter-assay variations (Fig. 2B). It is noteworthy that the correlation between measured and actual CCNA1 methylation percentages was not linear, but exponential.

CCNA1 methylation and expression in cervical tissues

The discovery of an inverse correlation between CCNA1 methylation and expression in CC lines suggested possibility of the same situation in vivo. To test this hypothesis, we evaluated the epigenetic control in vivo. Six frozen OTC-embedded CCs and five normal cervices were microdissected and subjected to duplex MSP and CCNA1 RT-PCR. Figure 3 shows examples of typical in vivo results. First, whereas no methylation could be observed, CCNA1 mRNA was discoverable by RT-PCR in normal cervix from both epithelium and connective tissue cells (Fig. 3A). In contrast, epigenetic control was detectable in cervical epithelia of CC patients from both malignant cells and adjacent histologically normal cervical epithelia. Nonetheless, in matched cases, a higher degree of methylation could be demonstrated in cancer than in normal cells. From all CCs, no CCNA1 mRNA was detectable. Interestingly, even if methylation was detected, CCNA1 was expressed in malignancy-adjacent histologically normal cervical tissues. Moreover, an inverse correlation between the methylation level and mRNA quantity was observed. CCNA1 expression in methylated malignancy-adjacent histologically normal cervical epithelium may be due to normal cell contamination or partial methylation at the promoter according to CC multistep progression. Whereas complete methylation could be observed in most cancer cells, partial and non-methylated CCNA1 was discovered in the adjacent epithelia (Fig. 3B). In conclusion, this experiment evaluating cervical tissue in vivo led to three conclusions. First, CCNA1 methylation was exclusively associated with cervical carcinogenesis. Second, the epigenetic alteration occurred earlier than morphological transformation of the cellular phenotype. Finally, methylation may play a role in this gene inactivation.

CCNA1 methylation incidence during multistep cervical carcinogenesis

Cervical intraepithelial neoplasia provides a crucial model to study the multistep process of carcinogenesis. Therefore, we evaluated the frequency of CCNA1 methylation in several cervical epithelial tissues with a distinctive degree of malignant transformation, normal cervix, CIN, microinvasive and CC, respectively. We selected 43, 25 and 30 cases of white blood cells (WBC), normal cervical biopsies and invasive CCs, respectively (Table 2). Among these samples, 13 WBC samples and 6 normal cervical samples, located at least 3 cm from the tumor margin and showing the absence of HPV DNA, originated from CC patients. For all cases, when a methylated amplicon was visible and the methylation percentage measured exceeded 5%, the test was deemed positive. All selected CCs were squamous and positive for HPV. Of the cases, 24 harbored HPV type 16, 4 had HPV type 18 and 2 cases displayed unclassifiable HPV types. Interestingly, a high frequency of methylation was exclusively present in CCs, i.e., 28 cases or 93.3% (Fig. 4A,B and Table 2). To reveal multistep carcinogenesis, we included 24 cases of SILs and 5 microinvasive cancers from exfoliated cervical cells. All cases were positive for oncogenic HPV, analyzed by Hybrid Capture 2. Whereas 60% and 36.6% of the microinvasive cancers and high SILs, respectively, demonstrated CCNA1 methylation, none of the HPV-associated low SILs exhibited these epigenetic changes (Fig. 4B and Table 2).

Histological characteristics	Total number of cases	CCNA1 promoter hypermethylation
		Absent	Present
WBC	43	43	0
Normal cervix	25	25	0
Low-grade SIL	13	13	0
High-grade SIL	11	7	4
Microinvasive cancer	5	2	3
Squamous cell CC	30	2	28
FIGO stage I-IIA	6	0	6
FIGO stage IIB-IV	24	2	22
Grade 1, keratinized type	9	0	9
Grade 2, non-keratinized type	21	2	19

FIGO, International Federation of Gynecology and Obstetrics.

This study demonstrated that: (i) CCNA1 promoter hypermethylation in HPV-associated squamous cell CC is unusually common; (ii) it is specific to CC; and (iii) the methylation is more common in invasive phenotypes compared to other histopathological stages during multistep carcinogenesis. This finding identifies both the interesting biology of CC and a potential clinical application of CCNA1 methylation as an additional molecular marker for the early diagnosis of invasive CC.

Annual cytology screening has dramatically increased the effectiveness of early CC detection. Nonetheless, additional tests will help to improve the sensitivity and specificity of a single Papanicolaou smear for histological analysis. Recently, testing for oncogenic HPVs has been introduced to aid in the triage of women with atypical squamous cells of undetermined significance (ASCUS). [28] However, because the majority of patients with HPV-associated lesions do not progress to invasive cancer, several studies have attempted to add a panel of tumor suppressor gene methylations to improve the effectiveness of molecular cytological diagnosis. [29, 30] Since the frequency of CCNA1 methylation is high and specific to invasive CC, this gene should be a good candidate to increase the coverage rate for early cancer detection.

In HNSCC, CCNA1 promoter hypermethylation is inversely related to TP53 mutation. [13] Nonetheless, the frequency of CCNA1 promoter hypermethylation in CC is high, whereas the function of TP53 in CC is usually impaired as a consequence of protein degradation induced by binding of the viral E6 protein. [18] This observation may be due to either differences in tissue types or pathophysiological outcomes of TP53 between mutations and diminution of the protein function subsequent to E6 binding. We prefer the latter hypothesis, since TP53 and CCNA1 have been shown to augment each other's expression. [13, 14] Consequently, the CCNA1 protein could help to increase physiologic TP53 to counter the function of E6, except for cases of TP53 mutation. In other words, alterations of both CCNA1 and TP53 in HNSCC will be redundant. In contrast, in CC, a decrease in CCNA1 protein should prevent the increment of TP53 that would have compensated for the protein destruction by E6.

Multistep process analysis revealed that CCNA1 methylation is remarkably specific for cervical carcinogenesis. The biological function of CCNA1 is to activate DNA breakage repair by mechanisms depending on CDK2 activity and Ku proteins. [14] It is interesting to hypothesize why the genomic instability, triggered by impairment of the CCNA1 function, is crucial as an early event in CC development. Perhaps the rate of spontaneous mutations in cervical epithelial cells is too low to accumulate sufficient malignancy-transformation-dependent oncogene and tumor suppressor gene mutations if the cells possess fully functional CCNA1. Therefore, the frequency of invasive CC devoid of CCNA1 methylation is limited.

This study demonstrates the strong association between CCNA1 promoter hypermethylation and invasive HPV-associated CC indicates that this gene could serve as an effective molecular marker. Moreover, our finding, in comparison with previous reports, [13, 14] also suggests that there is a possible molecular link between oncogenic HPVs, TP53 and CCNA1 promoter hypermethylation.