In the present work we have deciphered a new p63 pathway involving genes of cell-cell and cell-matrix adhesion in cervical carcinoma. It is not unexpected that genes modulating cell adhesion in epithelial cells were down-modulated in cervical carcinoma cells, thus participating in cell transformation. However, the mechanism by which this transcriptional modulation occurs is not yet known, and we can only speculate about potential mechanisms. Unlike p53, p63 status in cervical carcinoma as well as in other cancers is not well established, and it remains unclear whether p63 is a tumor suppressor gene or an oncogene. Point mutations of p63 are rare in cancers, and contradictory reports are present in the literature regarding its specific regulation in transformed cells, although it has been shown to be highly expressed in several epithelial cancers (reviewed in reference 13
). The complexity of the studies is due to the existence of multiple isoforms with opposite functions, with TAp63 as a likely tumor suppressor and ΔNp63 as an oncogene. In any case, p63 has not been shown previously to be degraded through the E6/E6AP proteasome pathway, and our results do not necessarily imply a direct involvement of this pathway in p63 modulation, although more experiments are needed to confirm that point. Another option is that modulation of the p63 target genes reflects modulation of the p63 transcription by p53, degradation of p53 leading to transcriptional down-regulation of p63, or, in the presence of E2, p53 stabilization leading to p63 activation.
The microarrays described here have confirmed and extended our previous observation that an important cluster of mitotic genes is modulated in cervical carcinoma. Furthermore, we could deduce that this specific modulation was essentially due to the expression of the E7 viral oncogene and the subsequent modulation of the pRb family of repressors of the E2F transcription factors. A recent study indicated that, indeed, repression of the E7/pRb pathway initiates induction of senescence in HeLa cells expressing the BPV1 E2 protein (20
). However, when mitotic genes were examined, no consensus E2F binding sequences could be found in their regulatory regions, contrasting with the genes involved in the G1
/S transition (12
). This means that other transcription factors are involved in transcriptional activation of the mitotic genes, which themselves are direct E2F target genes. Such transcription factors were found repressed by E2 in our experiments, including NF-YB in the present experiments (Table ) and B-Myb and FoxM1 in the previous microarray experiments (41
). The histone-like NF-Y transcription factor is the paradigm of a constitutive ubiquitous factor that prepares the promoter architecture for other factors to get access to it. It binds to the CCAAT box, which has been found in many cell cycle-regulated promoters (24
). In ChIP assays, NF-Y is found sequentially recruited to promoters of cell cycle genes together with E2F1 and E2F4 (4
). The B-Myb transcription factor is an E2F-regulated factor induced at the G1
/S transition, which plays a major role in activating G2
/M genes (47
). The FoxM1 transcription factor has been shown to activate a cluster of mitotic target genes, of which six are found modulated here: CENPA, cyclin B1, Nek2, Polo-like kinase, and the ubiquitin-conjugating enzyme 2C (22
). In addition, Aurora B, Survivin, CENPA, and cdc25B have been shown in ChIP assays to be direct targets of FoxM1 (45
). Modulation of mitotic genes in cervical carcinoma might therefore be essentially owing to modulation of the E2F target genes coding for the NF-Y, Myb, and FoxM1 transcription factors. In contrast, we showed here that the E6 pathway, through modulation of the p53 and p63 transcription factors, has only a minor influence on the mitotic gene regulation.
A recently published study identifies a proliferation gene cluster in cervical carcinoma associated with HPV16 or HPV18. The authors have reported 55 genes activated in cervical carcinoma that are related to the cell cycle, many of which are also E2F target genes. Interestingly, all these genes were also found modulated in our microarrays, indicating a strong overlap between the two studies (34
). Another very interesting convergence between the two studies is that, in both cases, the p53 target genes were not found modulated in biopsies of lesions, despite continuous expression of E6. This result may reflect the fact that p53 is not activated in normal cells in the absence of stress, thus inducing no marked difference whether E6 is expressed or not. Additionally, biopsies of cancer and normal tissues are heterogeneous, and modulation of the p53 target genes in E6-expressing cells may have been blurred by signals from contaminated cells, such as cells of the immune system or dermis fibroblasts. In any case, we deduce from our experiments that p53 target genes would be poor markers of carcinogenic progression of HPV-associated lesions of the cervix. In contrast, the large cluster of mitotic genes that were found modulated in established cell lines as well as in biopsies could be regarded as useful biomarkers of the evolution of lesions in cervical carcinoma.
Indeed, comparative analyses with published data indicated that, although E2F target genes of the G2
/M cell cycle transition are often found modulated in cancers, they do not form specifically large clusters as in cervical cancer. For example, a global study of the oncogenic pathway signatures in human cancers was recently published by the Bild et al., including an E2F pathway modulated through E2F3 expression in quiescent cells (2
). In this work the authors have deciphered five oncogenic pathways including E2F, RAS, MYC, SRC, and beta-cathenin. Strikingly, only 13 genes modulated in our arrays could be found among the genes specific to the E2F3 signature. We can deduce from these data and several comparative studies of other cancers that the HPV E7 signature is very specific and is biased in favor of mitotic gene activation.