Human papillomavirus (HPVs) are DNA viruses that produce hyperproliferative lesions in epithelial tissues. High-risk HPV genital infection, in particular HPV types 16 and 18, is associated with the development of genital cancer, while the low-risk HPVs induce benign genital warts. The oncogenic potential of HPV is, in part, mediated by the E6 and E7 proteins, which are known to bind and inactivate the p53 and retinoblastoma protein (pRb) respectively. Rb is one of the most investigated tumor suppressor genes and its dysregulation has been associated with neoplastic growth in a variety of tumors 1. The ability of HPV oncoproteins to disrupt growth regulatory proteins may have effects on the cyclin-dependent kinase (CDK) inhibitors linked to the G1- and G2- checkpoints. A group of CDK inhibitors, which includes p21, p27 and p57, inhibits the cyclin/cdk2, cdk4 complexes and causes G1 arrest. These proteins are rarely mutated in human tumors and their expression levels is of prognostic significance in a wide spectrum of tumors 2. P27 may be inactivated upon binding to DNA tumor virus oncoprotein, such as HPV 16 E7, can be deregulated early in carcinogenesis and may precede tumor invasion. Other HPV proteins act indirectly by altering the function of cellular factors. The loss of cell-cycle control leads to increased proliferation that can be detected by the increased number of cells in the cycle expressing specific markers 3.

Among the proteins and factors involved in cellular growth, VEGF (vascular endothelial growth factor) is a potent angiogenic factor that operates as an angiogenesis tumor mediator 4. The role of VEGF in cervical carcinogenesis has been studied and it is established that the VEGF expression has been related to microvascular density (MVD) in cervical intraepithelial neoplasia (CIN) and invasive carcinomas, suggesting that VEGF is an early marker of cervical carcinogenesis, with linearly increasing expression starting from low grade CIN 4. Another factor is eukaryotic translation initiation factors (Ets), which includes Elf, a lymphoid-specific member of this family, known to regulate inducible gene expression during T cell activation which includes increased angiogenesis. An over-expression of these factors can lead to neoplastic cellular change and tumor. Recent report supported a role of Elf-1 in the development of tumor angiogenesis and showed that targeting of Elf-1 in melanoma tumors resulted in decreased angiogenesis 5.

There is still relatively little data on biomarkers that may relate to increased risk of progression of CIN to cervical cancer 67 however, there is evidence that a few markers can be used as more specific risk assessment tools 8.

HIV/HPV co-infection is associated with a marked increase risk in progression of CIN to cervical cancer 9. Although the reasons for this observation are not well understood, cytokine profile alterations, including RANTES, have been described in co-infected patients 10.

Data suggests that HPV infection of the cervix might influence HIV pathogenesis by inducing both immune and inflammatory related protein production that enhances HIV expression 11.

Recent insights into molecular pathogenesis have demonstrated that HIV-1 proteins can directly lead to cancer growth by interfering with cellular functions. Also, HIV-1 proteins such as Tat can interact with the RB2/p130 tumor suppressor gene product and induce increased cell proliferation 12. However the mechanisms whereby HIV-1 subverts cell-cycle controls in the infected cervix have been unclear. Despite the fact that some reports have shown a role of VEGF, Rb, p27 in the HPV cervical CIN progression, little is known about the involvement and expression of these proteins in the HIV/HPV co-infection.

We investigated the hypothesis that HIV would modify the expression of regulatory and cell-cycle proteins in the cervix of HIV/HPV co-infected women. By using tissue micro-array (TMA) and immunohistochemistry to pRb, p27, VEGF and ELF-1, different CIN grades from HIV/HPV co-infected women were analyzed and compared to HPV/HIV-negative patients.

To our knowledge this is the first study to analyze the expression and protein alterations in different CIN grades in HIV and HPV co-infection correlating these protein alterations with histopathological and clinical features to evaluate putative prognostic marker potential.

Our study confirmed the hypothesis that there is significantly altered expression of regulatory and cell cycle proteins in the cervix due to HIV-1 infection occurring in the context of a co-existing HPV infection. Specifically, the epithelial cells expressing VEGF and p27 was significantly increased with HIV+/HPV+ co infection compared to HPV infection alone for both CIN I and CIN II/III lesions. This effect by HIV-1 was additive to the HPV infection, as the latter per se was also associated with statistically increased expression of VEGF and p27 in CIN I-III and invasive cancers. Some explanations could be raised, including immunosuppression and the persistent HPV infection frequently seen in the HIV-1 infected patients. However, the mechanisms and the pathway by which HIV-1 can trigger these proteins require further molecular studies.

The modulation of p27, VEGF, and, perhaps, Rb expression may provide some insights into why HIV-1 infection of the cervix appears to be associated with an increased risk of cervical cancer in women co-infected by HPV. HIV-1 proteins can directly promote cancer growth by interfering with cellular functions. For example, the HIV tat protein has been shown to physically interact with the RB2/p130 tumor suppressor gene product 12 which may promote the effect of HPV oncoproteins. The effect of the HPV oncoproteins E6 and E7 may, in turn, be related to viral load 16. On the other hand, the interactions between hr-HPV oncoproteins and molecules involved in cell cycle regulation are clearly related to the development of cervical cancer. Certainly other co-factors such as the HPV viral load may be predictive of the future risk of developing high-grade dysplasia or carcinoma, even in patients with negative Pap smears 16. In HIV-infected women, HPV viral load is consistently high, which may explain in part the higher probability of high-grade dysplasia development 17. In the present study we could not assay the local HPV viral load, but 57,5% of the HIV/HPV co-infected women exhibited high grade CIN. Persistent infection is the most important risk factor for initiating malignant transformation in the cervical epithelium, and a recent study reported that the timeframe from initial infection to pre-invasive and invasive disease appears to be at least 10–15 years 1.

Angiogenesis is important factor in the progression of CIN to invasive cancer and can be induced by many factors, including VEGF. In the present study we found that there was an increase in VEGF expression in the HPV only infected cervices, reaching the highest point in the invasive cancer cases, which is consistent with other report 4 that described a linear increasing from low grade CIN to high grade CIN and cancer lesions, suggesting that VEGF is a diagnostic marker for early stages of cervical carcinogenesis. However, in control cervices, we did find a low immunoreactivity both in the epithelium and stroma, perhaps explained by the fact that the vast majority of cervical tissues, even if taken from hysterectomy specimens where there is no suggestion of HPV infection, will exhibit inflammatory infiltrates in the cervix, that is, chronic cervicitis. In the HPV+/HIV+ co-infected cervices VEGF was highly expressed in both CIN I and CIN II/III, and showed no statistical difference.

The Elf-1 factor is a transcription factor that regulates a number of inducible lymphoid-specific genes and also in the development of tumor angiogenesis 5. Elf-1 expression was statistically equivalent in the HPV alone and HPV/HIV-1 co-infection, suggesting that HIV-1 per se was not modulating its expression. Elf-1 expression did increase in CIN II/III and carcinoma lesions compared to the negative controls in HPV alone infected cervices and did reach statistical significance, suggesting that molecular events in the more progressed HPV infected cervices was increasing Elf-1 expression. Interestingly, Elf-1 was less expressed in patients without ARV treatment compared with those undergoing mono, dual or HAART (p < 0.005), possibly justified by the fact that some cellular transcription factors can bind to the HIV-1 5' long terminal repeat (LTR). However, the contribution of these factors to human immunodeficiency virus type 1 replication in infected individuals remains obscure 18.

Many tumor suppressor genes, most of them involved in the cell cycle, have been described, the functional loss of these suppressor gene pathways probably being the basis for cervical carcinogenesis. P27, an important cell cycle inhibitor protein, is involved in the growth and differentiation of the normal squamous epithelia of the uterine cervix, and controversial data have been described in the literature. Some authors have found that low levels of p27 are associated with poor prognosis and suggested that decreased p27 protein might contribute to the altered kinetics of the dysplastic and neoplastic cervical epithelium 1920, whereas others have suggested that an increased and/or aberrant function of p27 expression may occur in invasive squamous cell carcinoma of the cervix 21. Our data showed a marked increase in p27 infection by HPV alone in all CIN grades compared to controls, and decreased in tumor compared to any other group of CIN. One possibility to explain this increase is the blocking of p27 activity by the HPV oncogene E7 which binds p27 via its C terminus. However, the mechanisms by which the HIV would modify this activity is unknown. It is important to recall that cell cycle control is a multifactorial system and, for example, decreased Rb expression may increase cyclin E and cdk2 which, in turn, can overcome the tumor suppressive effect of increased p27 22. In this connection, our data show that there was a 3-fold increase in Rb expression with HIV-1 co-infection versus HPV infection alone, but this did not reach statistical significance, there may well be a stimulatory effect on Rb production due to HIV-1 co-infection in this context. This possibility will require additional studies. Nonetheless, since Rb expression decreased in CIN II/III and carcinoma in HPV only infected cervices compared to the negative controls, corroborating the previously published data 2324. Interestingly, HIV-1 co-infection appeared to rescue Rb expression which may, in turn, potentiate the increased expression of p27 25. However, this interaction by itself may not be sufficient for neoplastic transformation and other cofactors may be required. Nevertheless, it is likely that the deregulation of cellular genes and function by Tat can also cause abnormalities that may contribute to AIDS pathogenesis and to the development of AIDS-associated disorders 12.

This comparative study adopting Tissue Micro-Array for possible putative prognostic markers, made possible the comparison of a large number of different CIN grade samples, and demonstrated to be a useful research tool, primarily for immunohistochemistry procedures, in spite of some limitations. First, we could not assay high risk-HPV sub-typing in all samples, which prevented comparison between Hr-HPV types with the expression markers in the different CIN grades. Second, we cannot accurately determine the HPV type in these relatively small cores. Third, we decided to use nonspecific cervicitis as controls, as most transformation zone biopsies, even from women with no evidence of HPV or HIV-1 infection, show nonspecific cervicitis. Despite the limitations, we were able to document statistically significant changes due to HIV-1 co-infection in HPV infected cervices.

The neoplastic process of the uterine cervix occurs in the epithelium. In the present study we analyzed the expression of different markers both in the epithelium and stromal tissue, however we focused our analysis in the epithelium. Although VEGF demonstrated significant differences (p < 0.001) in low grade CIN compared to control in the HPV+/HIV-negative patients, this marker was also increased in high grade CIN, suggesting that it is not a good marker for CIN progression. Increasing grades of CIN were associated with decreased Rb expression cervices, however further studies with more infected cervices should be done in order to confirm these observations. Additionally, we suggest that many immune markers that have been studied as CIN progressors should also be evaluated in HIV/HPV co-infection, since this group of patients may have different clinical and immune outcomes.

In conclusion, this study demonstrated a significant altered expression of regulatory and cell cycle proteins such as VEGF and p27 in cervical samples from HPV+/HIV+ co-infected compared to HPV+/HIV-negative women, suggesting that HIV-1 virus may be triggering these proteins which in turn could facilitate viral persistence and progression to an invasive tumor.

HPV: Human Papillomavirus; Rb: Retinoblastoma protein; VEGF: vascular endothelial growth factor; Elf: Eukariotic Translation Initiation Factors; TMA: Tissue Micro-Array; HIV: Human Immunodeficiency Syndrome; CIN: Cervical intra-epithelial neoplasia; ARV: antiretroviral; HAART: Highly active antiretroviral treatment; SCC: Squamous cell carcinoma; STD: Sexually Transmitted Disease.

The authors declare that they have no competing interests.

AFN conceived the study design, prepared the manuscript and was the main investigator. ARCP and SRS carried out the Tissue Micro-array studies and the immunohistochemistry analysis. GJN contributed to the immunohistochemical study and in the preparation of the manuscript. BG, AP and FR contributed toward the clinical patient selection and treatment. LSV participated in the statistical analysis, JRLS and CP participated in the discussion of the results and in the preparation of the manuscript. All authors have read and approved the final manuscript.