Therapeutic potential of an adenovirus expressing p73 beta, a p53 homologue, against human papilloma virus positive cervical cancer in vitro and in vivo

Human papilloma virus (HPV) infection is the most important risk factor for cervical cancer development. p53 based gene therapy is not suitable for cervical cancer because HPV oncoprotein E6 inactivates p53 protein by targeting it for ubiquitin mediated degradation. Here we evaluated the efficiency of Ad-p73, a replication deficient adenovirus expressing p73beta a p53 homologue, to inhibit the growth of HPV positive cervical cancer cells in vitro using tissue culture system and in vivo using human xenografts in nude mice. Ad-p73, but not Ad-p53 (p53 adenovirus), inhibited the growth in vitro of three different HPV positive cervical cancer cell lines, HeLa, ME180, and SiHa, efficiently, which correlated with stable expression of functional p73 protein. However, the growth of a HPV negative cervical cancer cell line, C33A, was inhibited equally by both Ad-p73 and Ad-p53. In addition, we show that Ad-p73 preinfected HeLa cells and HCT116 E6 cells, an E6 stable cell line, failed to form tumors in nude mice unlike Ad-p53 or Ad-LacZ preinfected cells. Moreover, Ad-p73, but not Ad-p53, inhibited completely the growth of already established tumors of HeLa or HCT116 E6 cells. Furthermore, the ability of p73 to inhibit the growth of these tumors correlated with the stable expression of p73 protein with the concomitant induction of its target gene p21(WAF1/CIP1) and induction of apoptosis in tumor cells. These results suggest that Ad-p73 inhibits efficiently the growth in vitro and tumorigenicity and tumor growth in vivo of HPV positive cervical cancer cells and that p73-based approach should be explored as a potential therapeutic model for the treatment of cervical cancer.     

Functional inactivation of p73, a homolog of p53 tumor suppressor protein, by human papillomavirus E6 proteins

Human papillomavirus (HPV) is strongly implicated as a causative agent in the etiology of cervical cancer. Of its gene products, E6 binds to and inactivates p53 tumor suppressor protein by ubiquitin/proteasome-dependent degradation. Recently, p73, a novel family of p53, has been identified and demonstrated, like p53, to activate p21(WAF1). Here we show that p73 is also inactivated by HPV-E6, but ubiquitin-mediated proteolysis is not responsive. Yeast two-hybrid and GST pull-down assays indicate a physical interaction between p73 and either HPV-16 or HPV-11 E6 proteins in vivo and in vitro, respectively. The transactivation domain (amino acid residues 1 to 49) is found to be absolutely required for the interaction. Transient co-expression of E6 significantly inhibits the p73-mdiated activation of p21(WAF1) promoter in a p53-defective C33A cell line. Using Gal4-p73 fusion protein, we demonstrate that E6 inhibition of p73 transactivation function is independent of sequence-specific DNA binding, which is confirmed by a direct electrophoretic mobility shift assay. Moreover, E6 inhibits p73 function by interfering with the activity of the amino-terminal activation domain. Co-transfection of E6 mutants reveals that the same portion of E6 appears to be responsible for the inactivation of p53 and p73 function. However, the inactivation mechanism of p73 is clearly different from that of p53, because p73, unlike p53, is inactivated by both high- and low-risk E6s and is not susceptible to E6-dependent proteolysis. These overall results, consequently, suggest that in addition to the inactivation of p53, the functional interference of p73 by HPV-E6 may, at least in part, contribute to E6-mediated transformation and hyperproliferation of cervical cells.     

Enhanced specificity of the p53 family proteins-based adenoviral gene therapy in uterine cervical cancer cells with E2F1-responsive promoters

p63 and p73, the p53 family proteins, are similar to p53 in many aspects: structural homology, transactivation of p53-downstream genes, and induction of apoptosis. Interestingly, they also differ from p53; in particular, they are not inhibited by viral oncoproteins such as HPV E6. This feature would be an advantage over p53 in HPV-associated cancers and therefore, we evaluated the therapeutic potentials of various p53 family proteins (p73alpha, p73beta, p63alpha and p63gamma) against HPV-infected cervical cancers. In clonogenic assay, exogenous expression of p73alpha, p73beta and p63gamma inhibited the colony formation of HPV-positive cervical cancer cells under G418- selection while p53 could not. Recombinant adenoviruses Ad/CMVp73alpha, Ad/CMVp73beta and Ad/CMVp63gamma induced potent apoptosis in all infected cervical cancers (CasKi, SiHa, HeLa, C33A, SNU682, SNU17, SNU1005, SNU703), irrespective of their HPV-infection status. Unfortunately however, Ad/CMVp73alpha, Ad/CMVp73beta, and Ad/CMVp63gamma inhibited also normal cells such as endothelial cells, fibroblasts, and keratinocytes thus, tumorspecific promoter was indispensable to the p53 family proteins-based therapy. Here we report a stringent tumor killing by p73beta in combination with ESM6, a synthetic promoter targeting the DNA tumor virusassociated cancers. Recombinant adenoviruses encoding p73beta by ESM6 (Ad/ESM6p73beta and Ad/ESM6p73bENH) expressed p73beta and induced apoptosis only in the cancer cells but not in normal cells. Collectively, we suggest that the p53 family proteins are potent therapeutic agents for HPV-associated uterine cervical cancers and ESM6 mediated expression of the p53 family proteins would be a safe and strong tumor targeting strategy.     

Induction of the p53-target gene GADD45 in HPV-positive cancer cells.

The E6 oncoprotein of human papillomaviruses (HPVs) has the potential to functionally antagonize p53. In several experimental model systems, ectopic expression of E6 can block the genotoxic induction of the growth inhibitory p53 target gene gadd45, suggesting that the inactivation of this pathway may play a major role for HPV-associated cell transformation. Here, we investigated whether this reflects the regulation of gadd45 expression in carcinoma-derived HPV-positive cells. We found that the gadd45 gene is efficiently induced by mitomycin C, cisplatin, and UV irradiation in a series of HPV-positive cervical cancer cell lines. Moreover, clear induction of gadd45 gene expression was also observed following treatment with gamma-irradiation, a pathway that is strictly dependent on functional p53. This contrasted with findings in human foreskin keratinocytes experimentally immortalized by expressing the HPV16 E6, E7, or E6/E7 oncogenes from the heterologous CMV promoter, where expression of the E6 gene was linked to a lack of gadd45 induction following gamma-irradiation. These results indicate (1) that the tumorigenic phenotype of HPV-positive cancer cells is not linked to an inability to induce the gadd45 gene following DNA damage, (2) that experimental model systems in which the E6 gene is expressed ectopically and/or in a different cellular context do not necessarily reflect the regulation of p53-associated pathways in HPV-positive cancer cells and (3) that a pathway strictly depending on functional p53 is inducible in HPV-positive cancer cells, providing direct evidence that the endogenous p53 protein in these cells is competent to activate a cellular target gene, despite coexpression of the viral E6 oncogene.     

Proteasome inhibition mediates p53 reactivation and anti-cancer activity of 6-Gingerol in cervical cancer cells

Human papilloma virus (HPV) expressing E6 and E7 oncoproteins, is known to inactivate the tumor suppressor p53 through proteasomal degradation in cervical cancers. Therefore, use of small molecules for inhibition of proteasome function and induction of p53 reactivation is a promising strategy for induction of apoptosis in cervical cancer cells. The polyphenolic alkanone, 6-Gingerol (6G), present in the pungent extracts of ginger (Zingiber officinale Roscoe) has shown potent anti-tumorigenic and pro-apoptotic activities against a variety of cancers. In this study we explored the molecular mechanism of action of 6G in human cervical cancer cells in vitro and in vivo. 6G potently inhibited proliferation of the HPV positive cervical cancer cells. 6G was found to: (i) inhibit the chymotrypsin activity of proteasomes, (ii) induce reactivation of p53, (iii) increase levels of p21, (iv) induce DNA damage and G2/M cell cycle arrest, (v) alter expression levels of p53-associated apoptotic markers like, cleaved caspase-3 and PARP, and (vi) potentiate the cytotoxicity of cisplatin. 6G treatment induced significant reduction of tumor volume, tumor weight, proteasome inhibition and p53 accumulation in HeLa xenograft tumor cells in vivo. The 6G treatment was devoid of toxic effects as it did not affect body weights, hematological and osteogenic parameters. Taken together, our data underscores the therapeutic and chemosensitizing effects of 6G in the management and treatment of cervical cancer.     

HPV-16 E6 upregulation of DNMT1 through repression of tumor suppressor p53

The HPV-16 early proteins E6 and E7 are considered to function as oncoproteins in cervical cancer. DNA methyltransferase 1 (DNMT1) is one of the enzymes involved in epigenetic silencing of tumor suppressor genes. In the present study, the functional role and regulation of DNMT1 in HPV-16 E6 associated cervical cancer development were examined. Knockdown of E6 in HPV-16 positive human cervical carcinoma SiHa and CaSki cells led to the increase in p53, repression of DNMT1 protein and promoter activity. Moreover, p53 knockdown increased the DNMT1 protein as well as promoter activity, indicating that p53 may mediate E6 upregulation of DNMT1. In addition, E6 knockdown induced growth retardation in SiHa cells, and the effect was partially reverted by DNMT1 overexpression. The results suggest that HPV-16 E6 may act through p53/ DNMT1 to regulate the development of cervical cancer.     

HPV-16 E6 and E7 oncogene expression is downregulated as a result of Mdm2 knockdown

The carcinogenic potential of HPV infections is based on the integration and constitutive expression of the E6 and E7 genes which inhibit the p53 and Rb tumor suppressor proteins. In normal cells, Mdm2 regulates p53 in a negative feedback loop, and although Mdm2 is apparently functional in HPV-infected cells, E6 is the protein responsible for repressing p53 replacing Mdm2 function. The role of Mdm2 in HPV-positive cells is still elusive. In this study, Mdm2 was knocked down in an HPV-positive cervical cancer cell line; as a result we found downregulation of the expression of E6 and E7 and p53 upregulation.     

Upregulation of p18Ink4c expression by oncogenic HPV E6 via p53-miR-34a pathway

Binding of p53 to miR-34a promoter activates the expression of tumor-suppressive miR-34a. Oncogenic human papillomavirus (HPV) infection downregulates miR-34a expression through viral E6 degradation of p53. In our report, we found that miR-34a specifically targets p18Ink4c, a CDK4 and CDK6 inhibitor induced by E2F transactivation. HPV18(+) HeLa cells with ectopic miR-34a expression or by E6 siRNA knockdown-induced expression of endogenous miR-34a exhibited a substantial reduction of p18Ink4c in a dose-dependent manner, but had no effect on p16Ink4a, another member of CDK4/6 inhibitor family. In contrast, de novo infection by oncogenic HPVs of human keratinocyte-derived raft tissues increased p18Ink4c expression. Suppression of endogenous miR-34a in cell lines with a miR-34a inhibitor also increased p18Ink4c. We found that miR-34a suppresses the expression of p18Ink4c by binding to a specific seed match in the 5' UTR of p18Ink4c. Further investigation found remarkable increase of p18Ink4c in cervical precancer lesions and cervical cancer. Immunohistochemical staining of cervical tissue arrays showed increased expression of p18Ink4c in 68% of cervical cancer, 8.3% of chronic cervical inflammation and 4.8% of normal cervix. Although p18Ink4c inhibits cell proliferation in general and regulates E2F1 expression in HCT116 cells, it appears not to function as a tumor suppressor in cervical cancer cells lacking an intact G1 checkpoint because of viral E7 degradation of pRB. In summary, our study demonstrates an intimate connection among oncogenic HPV E6, p53, miR-34a and p18Ink4c and identifies p18Ink4c as a possible biomarker for cervical cancer.     

Inhibition of cervical cancer cell growth in vitro and in vivo with lentiviral-vector delivered short hairpin RNA targeting human papillomavirus E6 and E7 oncogenes

In this study, we investigated the suppressive effect of a short hairpin RNA delivered by a lentiviral vector (LV-shRNA) against human papillomavirus (HPV) type 18 E6 on the expression of the oncogenes E6 and E7 in cervical cancer HeLa cells both in vitro and in vivo. The LV-shRNA effectively delivered the shRNA to HeLa cells and lead to a dose-dependent reduction of E7 protein and the stabilization of E6 target proteins, p53 and p21. Low-dose infection of HeLa cells with LV-shRNA caused reduced cell growth and the induction of senescence, whereas a high-dose infection resulted in specific cell death via apoptosis. Transplant of HeLa cells infected with a low dose of LV-shRNA into Rag-/- mice significantly reduced the tumor weight, whereas transplant of cells infected with a high dose resulted in a complete loss of tumor growth. Systemic delivery of LV-shRNA into mice with established HeLa cell lung metastases led to a significant reduction in the number of tumor nodules. Our data collectively suggest that lentiviral delivery is an effective way to achieve stable suppression of E6/E7 oncogene expression and induce inhibition of tumor growth both in vitro and in vivo. These results encourage further investigation of this form of RNA interference as a promising treatment for cervical cancer.     

GFP/HPV-16E6 fusion protein induces apoptosis in MCF-7 and 293T cells using a transient expression system

Since mucosal high-risk human papillomavirus (HPV) E6 can target and degrade the tumor suppressor p53, it is recognized as a major causative agent of cervical cancer. However, to date the distribution of high-risk HPV-E6 protein remains elusive. Thus, in the present study we used a mammalian green fluorescent protein (GFP) expression system to express a GFP/HPV-16E6 fusion protein (GFP-16E6) in wild-type (wt) p53 cells, such as MCF-7 and 293T cells to investigate the trafficking and localization of E6 and p53. Following transfection, we observed that the overexpressed GFP-16E6 was a nuclear protein, and that endogenous wt p53 localized to the nucleus together with GFP-16E6. Strikingly, p53 levels were not decreased but increased in 24?h transfected with pGFP-16E6. Furthermore, we observed significant apoptosis induced by GFP-16E6, which proved to be dependent on p53 expression.     

Chemoradiation of cervical cancer cells: targeting human papillomavirus E6 and p53 leads to either augmented or attenuated apoptosis depending on the platinum carrier ligand

Recent clinical trials comparing concurrent chemotherapy and radiation with radiation alone in cervical cancer have shown that chemoradiation reduces the risk of death by 30-50%. Despite the clinical success, treatment responses at the cellular level are still inadequately explored. A key event in cervical carcinogenesis is the disruption of p53 tumor suppressor pathway by human papillomavirus (HPV) E6 oncogene. We found that regardless of the HPV type in SiHa (HPV 16+) CaSki (HPV 16+), HeLa (HPV 18+), and UT-DEC-1 (HPV 33+) cell lines, cisplatin, carboplatin, and a novel platinum compound, oxaliplatin, activated a p53 reporter and reduced the HPV E6 mRNA. Carboplatin and oxaliplatin treatment led also to stabilization of p53, whereas none of the platinums changed p73 levels. After irradiation (IR) alone, a decrease in HPV E6 mRNA levels and an activation of the p53-reporter were detected in SiHa, CaSki, and HeLa cells, but not in UT-DEC-1 cells. Concomitant platinum treatment and IR led to poly(ADP-ribose) polymerase cleavage as a sign of caspase-3 activation and apoptosis. Clonogenic survival was enhanced by expressing a dominant negative p53 or ectopic HPV16 E6 in SiHa and HeLa cells treated with IR, carboplatin, or oxaliplatin or with a combination of IR + carboplatin or oxaliplatin. In contrast, dominant negative p53 or ectopic HPV 16 E6 sensitized the cells to cisplatin. Pt chemotherapeutics and radiation had a synergistic cytotoxic effect as determined by Bliss independence criterion. Taken together, p53 has a significant role in the cellular response to chemoradiation treatment in cervical cancer cell lines, but p53 activity may have a dramatically different effect on cell survival depending on the platinum carrier ligand.     

Grape seed proanthocyanidins induce apoptosis through p53, Bax, and caspase 3 pathways

Grape seed proanthocyanidins (GSP) have been shown to inhibit skin chemical carcinogenesis and photocarcinogenesis in mice. The mechanisms responsible for the anticarcinogenic effects of GSP are not clearly understood. Here, we report that treatment of JB6 C141 cells (a well-developed cell culture model for studying tumor promotion in keratinocytes) and p53+/+ fibroblasts with GSP resulted in a dose-dependent induction of apoptosis. GSP-induced (20-80 g/ml) apoptosis was observed by using immunofluorescence (27-90% apoptosis) and flow cytometry (18-87% apoptosis). The induction of apoptosis by GSP was p53-dependent because it occurred mainly in cells expressing wild-type p53 (p53+/+; 15-80%) to a much greater extent than in p53-deficient cells (p53-/-; 6-20%). GSP-induced apoptosis in JB6 C141 cells was associated with increased expression of the tumor-suppressor protein, p53, and its phosphorylation at Ser15. The antiapoptotic proteins, Bcl-2 and Bcl-xl, were downregulated by GSP, whereas the expression of the pro-apoptotic protein, Bax, and the levels of cytochrome c release, Apaf-1, caspase-9, and cleaved caspase 3 (p19 and p17) were markedly increased in JB6 C141 cells. The downregulation of Bcl-2 and upregulation of Bax were also observed in wild-type p53 (p53+/+) fibroblasts but was not observed in their p53-deficient counterparts. These data clearly demonstrate that GSP-induced apoptosis is p53-dependent and mediated through the Bcl-2, Bax, and caspase 3 pathways.     

HPV16 E6-mediated stabilization of ErbB2 in neoplastic transformation of human cervical keratinocytes

Whether ErbB2 receptor tyrosine kinase contributes to cervical cancer is controversial. We have examined the effects of E6 and E7 genes of human papillomaviruses type 16 (HPV-16) on ErbB2 expression in primary human cervical keratinocytes (HCK) immortalized with hTERT (HCK1T). In E6-positive cells (HCK1T-E6 and HCK1T-E6E7), ErbB2 expression levels increased with the cell density. HCK1T-E6E7 showed impaired contact inhibition and anchorage-independent growth in soft agar which were abrogated with introduction of ErbB2-specific short hairpin RNA (shRNA) or an ErbB2 specific inhibitor AG825. Furthermore, increased ErbB2 expression was also observed in HPV16 positive cervical cancer cell lines and this was diminished by introduction of HPV16E6- or E6AP-shRNA. At post-confluence cell densities, ErbB2 protein was stabilized in the presence of E6 whereas increased ErbB2 expression was not obvious with E6 mutants incapable of degrading p53. Furthermore, introduction of p53-shRNA to HCK1T resulted in increased ErbB2 protein stability, indicating possible ErbB2 regulation through p53. Finally, we showed that tumor formation of ErbB2-shRNA introduced SiHa cells were almost abolished. Taken together, these data indicate an important role of ErbB2 regulation by HPV16 E6 in oncogenic transformation of human cervical keratinocytes.