An in vitro multistep carcinogenesis model for human cervical cancer

Human papillomaviruses (HPV) are believed to be the primary causal agents for development of cervical cancer, and deregulated expression of two viral oncogenes E6 and E7 in basal cells, mostly by integration, is considered to be a critical event for disease progression. However, lines of evidence suggest that, besides expression of E6 and E7 genes, additional host genetic alterations are required for cancer development. To directly test this hypothesis, we first transduced HPV16 E6 and E7 with or without hTERT into several lines of normal human cervical keratinocytes (HCK) from independent donors and then searched for additional alterations required for carcinogenesis. Oncogenic Hras(G12V) (Hras) provided marked tumor forming ability in nude mice and ErbB2 or c-Myc (Myc) endowed weaker but significant tumor forming ability. Combined transduction of Myc and Hras to HCKs expressing E6 and E7 resulted in the creation of highly potent tumor-initiating cells. These results show that only one or two genetic changes occurring after deregulated expression of high-risk HPV oncogenes might be sufficient for development of cervical cancer.     

Effect of human papillomavirus 16 oncoproteins on oncostatin M upregulation in oral squamous cell carcinoma

Human papillomavirus (HPV) infection modulates several host cytokines contributing to cancer development. Oncostatin M (OSM), an IL-6 family cytokine, acts to promote cell senescence and inhibit growth. Its dysregulation promotes cell survival, cell proliferation and metastasis in various malignancies. The effect of HPV on OSM dysregulation has not been investigated. To elucidate this, immunohistochemistry was used on formalin-fixed, paraffin-embedded oral squamous cell carcinoma (OSCC) tissues: HPV-positive (50) and HPV-negative (50) cases. Immortalized human cervical keratinocytes expressing HPV16E6 (HCK1T, Tet-On system) were used to demonstrate the role of HPV16E6 in OSM expression. In addition, a vector containing HPV16E6/E7 was transiently transfected into oral cancer cell lines. Cell viability, cell-cycle progression and cell migration were evaluated using flow cytometry and a wound healing assay, respectively. The results showed various intensities of OSM expression in OSCC. Interestingly, the median percentages of strongly stained cells were significantly higher in HPV-positive OSCCs than in HPV-negative OSCCs. To explore the role of HPV oncoproteins on OSM expression, the expression of HPV16E6 in the HCK1T Tet-On condition was induced by doxycycline and HPV16E6 was found to significantly upregulate levels of OSM mRNA and protein, with concomitant upregulation of c-Myc. In addition, the levels of OSM mRNA and protein in E6/E7 transiently transfected oral cancer cells also gradually increased in a time-dependent manner and these transfected cells showed greater viability and higher migration rates and cell-cycle progression than controls. This result demonstrates that HPV16 oncoproteins upregulate OSM and play an important role to promote OSCC development.     

Activities of E6 Protein of Human Papillomavirus 16 Asian Variant on miR-21 Up-regulation and Expression of Human Immune Response Genes

Background: Variants of human papillomavirus (HPV) show more oncogenicity than do prototypes. TheHPV16 Asian variant (HPV16As) plays a major role in cervical cancer of Asian populations. Some amino acidchanges in the E6 protein of HPV16 variants affect E6 functions such as p53 interaction and host immunesurveillance. This study aimed to investigate activities of HPV16As E6 protein on modulation of expressionof miRNA-21 as well as interferon regulatory factors (IRFs) 1, 3, 7 and c-fos. Materials and Methods: Vectorsexpressing E6 protein of HPV16As (E6D25E) or HPV16 prototype (E6Pro) were constructed and transfected intoC33A cells. HCK1T cells expressing E6D25E or E6Pro were established by transducing retrovirus-containingE6D25E or 16E6Pro. The E6AP-binding activity of E6 and proliferation of the transfected C33A cells weredetermined. MiR-21 and mRNA of interesting genes were detected in the transfected C33A cells and/or theHCK1T cells, with or without treatment by culture medium from HeLa cells (HeLa-CM). Results: E6D25Eshowed binding activity with E6AP similar to that of E6Pro. Interestingly, E6D25E showed a higher activity ofmiR-21 induction than did E6Pro in C33A cells expressing E6 protein. This result was similar to the HCK1Tcells expressing E6 protein, with HeLa-CM treatment. The miR-21 up-regulation significantly corresponded toits target expression. Different levels of expression of IRFs were also observed in the HCK1T cells expressing E6protein. Interestingly, when treated with HeLa-CM, IRFs 1, 3 and 7 as well as c-fos were significantly suppressedin the HCK1T cells expressing E6D25E, whereas those in the HCK1T cells expressing E6Pro were induced. Asimilar situation was seen for IFN-α and IFN-β. Conclusions: E6D25E of the HPV16As variant differed fromthe E6 prototype in its activities on epigenetic modulation and immune surveillance and this might be a keyfactor for the important role of this variant in cervical cancer progression.     

Expression of human papillomavirus type 16 E6 and E7 oncoproteins in primary foreskin keratinocytes is sufficient to alter the expression of angiogenic factors

Human papillomavirus (HPV) 16 is involved in causing cervical cancer. The E6 and E7 proteins of HPV 16 immortalize human keratinocytes and this is due, at least in part, to inactivation of the tumor suppressor proteins p53 and pRB. These tumor suppressor proteins also regulate the expression of pro- and antiangiogenic factors by cells. For this reason, experiments were conducted to determine whether the expression of E6 and E7 in primary keratinocytes alters the phenotype of these cells such that they express diminished levels of antiangiogenic factors and/or increased levels of proangiogenic factors. To avoid variances in experimental observations, pools of human foreskin keratinocytes from multiple sources were infected with recombinant retrovirus expressing HPV 16 E6 and E7 or control retrovirus. Gene array analysis, RT-PCR, ELISAs and Western blotting showed that in cells expressing HPV 16 E6 and E7, expression levels of two potent angiogenesis inhibitors, thrombospondin-1 and maspin, were lower compared to controls. Additionally, major angiogenesis inducers, interleukin-8 and vascular endothelial growth factor (VEGF), were increased relative to controls. VEGF can be produced as multiple splice variants, all of which are required for the formation of patent blood vessels. The expression of HPV 16 E6 and E7 in keratinocytes augmented expression of VEGF 121, 145, 165 and 189. These observations show that HPV 16 E6 and E7 alter the phenotype of primary keratinocytes, diminishing expression of inhibitors and increasing expression of inducers of angiogenesis. This altered phenotype may permit keratinocytes infected by HPV 16 to play a role in the progression of cancer by permitting tumors to acquire a blood supply permissive of growth and spread.     

Immortalization of oral keratinocytes by functional inactivation of the p53 and pRb pathways

A subgroup of head and neck squamous cell carcinomas (HNSCCs) contains high‐risk human papillomavirus‐type 16 (HPV16). The viral E6 and E7 oncoproteins inactivate the p53 and pRb proteins, respectively. We examined the causative effect of HPV16 E6 and E7 expression on the immortalization of normal oral keratinocytes (OKCs) and compared the resulting phenotype with alternative ways of p53‐ and pRb‐pathway abrogation frequently found in HNSCCs without HPV. Primary OKCs were conditionally immortalized with temperature‐sensitive SV40 large T‐antigen and human telomerase, allowing these cells to return to their senescent primary state after temperature shift. HPV16 E6 and E7 were introduced to overcome senescence, determined with population doubling (PD) as read‐out. For comparison, we downregulated p53 and p16 by short hairpin RNA genes and expressed mutant p53R(175)H and cyclinD1. Expression of HPV16 E6 caused an extended life span similar to expression of mutant p53R(175)H or p53 knockdown. Expression of mutant p53R(175)H seemed to cause additional activation of the hypoxia and WNT signaling pathways. HPV16 E7 expression had no direct effect on lifespan, similar to p16 knockdown or cyclinD1 expression. In combination with HPV16 E6 or other functional inactivations of p53, abrogation of the pRb‐pathway by either HPV16 E7 or other manipulations caused an immortal phenotype. Our data show the causative role of HPV16 E6/E7 in early squamous carcinogenesis. Activity of each gene could be mimicked by other genetic events frequently found in HNSCC without HPV. This data provides the experimental proof of causal association of HPV in HNSCC carcinogenesis and further support the crucial role of the p53‐ and pRb‐pathways.     

E7 proteins from oncogenic human papillomavirus types transactivate p73: role in cervical intraepithelial neoplasia

In common with other E2F1 responsive genes such as p14(ARF) and B-myb, the promoter of p73 is shown to be positively regulated in cell lines and primary human keratinocytes by E7 proteins from oncogenic human papillomavirus (HPV) types 16, 18, 31 and 33, but not HPV 6. Mutational analysis revealed that transactivation of the p73 promoter by HPV 16E7 requires association with pRb. Expression of p73 in normal cervical epithelium is confined to the basal and supra-basal layers. In contrast, expression in neoplastic lesions is detected throughout the epithelium and increases with grade of neoplasia, being maximal in squamous cell cancers (SCC). Deregulation of expression of the N-terminal splice variant p73Delta2 was observed in a significant proportion of cancers, but not in normal epithelium. The frequent over-expression of p73Delta2, which has recognized transdominant properties, in malignant and pre-malignant lesions suggests a role in the oncogenic process in cervical epithelium.     

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.     

NF‐κ B‐dependent upregulation of ICAM‐1 by HPV16‐E6/E7 facilitates NK cell/target cell interaction

NK cell recognition of tumor cells is mediated by a delicate balance of signals received by MHC class I‐binding inhibitory NK cell receptors and activating NK cell receptors, which mainly bind to virus‐, stress‐ or tumor‐induced ligands. In addition, adhesion molecules such as the intercellular adhesion molecule‐1 (ICAM‐1) and its receptors, the lymphocyte function‐associated antigen‐1 (LFA‐1) and Mac‐1, are crucial for immune synapse formation and NK cell‐mediated killing. In this study, we show that expression of the adhesion molecule ICAM‐1 was rapidly induced by E6 and ‐E7 oncoproteins of HPV16, ‐18, ‐5 and ‐8, but not of HPV38 and ‐6 in primary human keratinocytes after retroviral transduction. ICAM‐1 was upregulated in E6E7‐expressing keratinocytes both at mRNA and protein levels. The observed ICAM‐1 upregulation in HPV16‐E6E7‐expressing keratinocytes was partially dependent on activation of the NF‐κB pathway. Importantly, the upregulated ICAM‐1 expression in HPV16‐E6E7‐expressing keratinocytes led to enhanced conjugate formation with NK cells. We previously showed that HPV16‐positive cervical carcinomas frequently express low levels of inhibitory NK cell ligands and high levels of activating NK cell ligands. Moreover, levels of the adhesion molecule ICAM‐1 are enhanced by HPV16‐E6/E7. Therefore, strategies that aim at harnessing NK cells might be beneficial for the treatment of cervical carcinoma.