Title: Cidofovir as an antiproliferative agent: novel insights into the activity against human papillomavirus-positive cervical carcinoma cells
Other Titles: Cidofovir als antiproliferatief agens: nieuwe inzichten in de activiteit tegen humaan papillomavirus-positieve baarmoederhalskanker cellen
Authors: De Schutter, Tim
Issue Date: 26-Jun-2013
Abstract: Human papillomaviruses (HPVs) belong to the Papillomaviridae, a family of small non-enveloped double-stranded DNA viruses. All papillomaviruses have a strong epithelial tropism and can induce cell proliferation in skin or mucosa. Cutaneous HPVs generally cause benign lesions, whereas mucosal types are subdivided in low-risk and high-risk types based on their capacity to induce either benign or malignant lesions. The main hallmark of HPV is the causal connection with cervical carcinoma, the second most common cancer among women worldwide. Virtually all cases of cervical cancer are attributable to infection with high-risk HPVs, types 16 and 18 accounting for more than 70% of all cases. Moreover, fractions of various other cancers, in particular cancer of the penis, vulva, vagina, anus, and oropharynx, have also been associated with high-risk HPV infection.To date, no specific therapies are available for the treatment of HPV-induced lesions, and surgical removal of the transformed tissue remains the main option for treatment of HPV infections. Two prophylactic HPV vaccines are currently available and represent a major advance in the prevention of HPV-related (cervical) malignancies. However, these vaccines do not offer protection against all HPV types and lack any therapeutic effect. Hence, there is a clear need for the development of novel therapeutic strategies for HPV-associated diseases, in particular for immunosuppressed patients that are more susceptible to persistent and widespread HPV infections, as well as a large proportion of the population that will remain unvaccinated for the foreseeable future.Several studies have shown the potential use of the nucleotide analogue cidofovir (CDV) to treat HPV infections by either topical administration or local injection. However, the mechanism underlying the activity of CDV against HPV-induced proliferation remains to be elucidated. Therefore, the aim of this project was on the one hand to study the mechanism of action of CDV and its selectivity for HPV-transformed cells, and on the other hand to characterize HPV-positive cells that have been selected for resistance against CDV.In the second chapter, we studied the effect of CDV exposure on gene expression in HPV-positive cervical carcinoma cells, HPV-negative tumor cells, and normal keratinocytes by means of full human genome microarrays. These experiments allowed the identification of cellular genes involved in the responses to CDV in different cell types. Our data confirmed that CDV induces apoptosis in HPV-positive tumor cells. Moreover, CDV was shown to be incorporated in newly synthesized DNA strands, causing DNA damage. In contrast to tumor cells that are typically deficient in DNA damage repair mechanisms, normal keratinocytes could activate DNA damage signaling pathways, cell cycle regulation, and initiate DNA damage repair by homologous recombination. In HPV-transformed cells, the activities of the viral oncoproteins E6 and E7 are known to impede these DNA repair mechanisms, causing accumulation of DNA damage that leads to induction of apoptosis. Cell cycle progression was inhibited in the HPV-negative tumor cells and no DNA repair pathways were activated, also resulting in apoptosis in these cells. Although an effect on inflammatory response was seen in all cell types, different pathways were identified in normal keratinocytes compared to malignant cells. Notably, acute phase response signaling was exclusively activated in malignant cells. In addition, several other pathways and biological functions were found to be induced or repressed differently by CDV in normal keratinocytes and tumor cells.The third chapter describes the effect of CDV treatment on an HPV16-positive cervical carcinoma xenograft in athymic nude mice. We demonstrated that intratumoral, but not systemic, CDV treatment not only successfully suppressed tumor growth in this model, but also ameliorated the pathology associated with this xenograft. The treatment afforded a beneficial effect on body weight gain and a reduction in splenomegaly in tumor-bearing mice. Furthermore, the treatment was able to weaken the inflammatory state induced by the xenograft, as evidenced by diminished host- and tumor-derived cytokine serum levels and by a reduction in the number of inflammatory cells (primarily neutrophils) in the spleen and infiltrations of these cells in distant organs (i.e. liver and lungs). Administration of CDV to tumor-free animals did not have a direct effect on these parameters.In the fourth chapter, we described CDV-resistant sublines that have been established by subculturing cells in increasing concentrations of the compound. In vitro phenotyping of these cells showed a more than 100-fold increase in resistance to CDV as well as a slower growth compared to the parental cells. Although these cells demonstrated an increased resistance to structurally related compounds, no marked cross-resistance to other chemotherapeutics was observed with the exception of low levels of resistance to fludarabine. Multi-drug resistance, a mechanism that is frequently associated with drug resistance in cancer, did not appear to be involved in CDV-resistance in these sublines. Moreover, genotyping of these CDV-resistant cells did not reveal any mutations in cellular DNA polymerases that could explain the resistant phenotype. Comparative microarray analysis of CDV-resistant cells and parental cells highlighted several biological processes and pathways to be associated with acquisition of resistance, indicating that several factors contribute to the resistant phenotype in a single subline, and that different mechanisms were involved in the CDV-resistant phenotype of distinct sublines. Thus, our in vitro characterization of CDV-resistance suggested that acquisition of resistance to CDV in (HPV-positive) tumor cells is a multifactorial process.The alterations in expression levels of genes involved in inflammation in CDV-resistant HPV16-positive tumor cells in comparison with parental cells were further analyzed in chapter five. The hypothesis that acquisition of resistance to CDV inthese cells was associated with an effect on inflammation was investigated in the athymic nude mice xenograft model. The CDV-resistant cells exhibited a slower tumor growth in vivo, consistent with the decreased growth rate in vitro. In addition, we observed a reduced pathogenicity of the resistant cells, as evidenced by the lack of splenomegaly, lower immune cell populations in the spleen (in particular neutrophils), and reduced host- and tumor-derived cytokine levels in the serum in comparison with the parental cells.In conclusion, we demonstrated the mechanism by which CDV induces apoptosis and the basis for its selectivity for HPV-positive tumor cells. Characterization of the CDV-resistant sublines revealed multiple factors playing a role in the acquisition of resistance. Furthermore, CDV treatment was also found to affect the inflammatory response and development of CDV-resistance was associated with reduced pathogenicity in vivo. Taken together, our findings underline the clinical potential of CDV in the treatment of HPV-induced diseases.
Publication status: published
KU Leuven publication type: TH
Appears in Collections:Laboratory of Virology and Chemotherapy (Rega Institute)

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