Cervical carcinomas result from cellular transformation by the human papillomavirus (HPV) E6 and E7 oncogenes which are constitutively expressed in cancer cells. The E6 oncogene degrades p53 thereby modulating a large set of p53 target genes as shown previously in the cervical carcinoma cell line HeLa. Here we show that the TAp63β isoform of the p63 transcription factor is also a target of E6. The p63 gene plays an essential role in skin homeostasis and is expressed as at least six isoforms. One of these isoforms, ΔNp63α, has been found overexpressed in squamous cell carcinomas and is shown here to be constitutively expressed in Caski cells associated with HPV16. We therefore explored the role of p63 in these cells by performing microarray analyses after repression of endogenous E6/E7 expression. Upon repression of the oncogenes, a large set of p53 target genes was found activated together with many p63 target genes related to cell adhesion. However, through siRNA silencing and ectopic expression of various p63 isoforms we demonstrated that TAp63β is involved in activation of this cell adhesion pathway instead of the constitutively expressed ΔNp63α and β. Furthermore, we showed in cotransfection experiments, combined with E6AP siRNA silencing, that E6 induces an accelerated degradation of TAp63β although not through the E6AP ubiquitin ligase used for degradation of p53. Repression of E6 transcription also induces stabilization of endogenous TAp63β in cervical carcinoma cells that lead to an increased concentration of focal adhesions at the cell surface. Consequently, TAp63β is the only p63 isoform suppressed by E6 in cervical carcinoma as demonstrated previously for p53. Down-modulation of focal adhesions through disruption of TAp63β therefore appears as a novel E6-dependent pathway in transformation. These findings identify a major physiological role for TAp63β in anchorage independent growth that might represent a new critical pathway in human carcinogenesis.
High-risk human papillomavirus infection can cause cancer of the uterine cervix. The viral proteins leading to transformation of the infected keratinocytes are the E6 and E7 oncogenes which interact with and induce degradation of the cell cycle regulators p53 and pRB. In cervical carcinoma cells, repression of E6/E7 stabilizes the p53 transcription factor leading to activation of a large group of cellular p53 target genes. Here we show that repression of E6/E7 also induces transcriptional activation of an additional large set of genes involved in cell adhesion including previously described p63 target genes. Indeed, we further demonstrated that these p63 target genes are activated by TAp63β and not by p53 or by the ΔNp63α or β isoforms, even though these transcription factors are also expressed in these cells. In cervical carcinoma cells, E6 expression therefore leads to TAp63β degradation thereby allowing anchorage independent growth. Our work describes a new E6-dependent transformation pathway in HPV-associated carcinogenesis. TAp63β inhibition may also represent a common pathway to activate anchorage independent growth in cancers.
Specific types of high risk Human papillomaviruses (HR-HPVs) particularly, HPV types 16 and 18 cause cervical cancer and while the two recently developed vaccines against these HPV types are prophylactic in nature, therapeutic options for treatment and management of already existing HPV infection are not available as yet. Because transcription factor, Activator Protein-1 (AP-1) plays a central role in HPV-mediated cervical carcinogenesis, we explored the possibility of its therapeutic targeting by berberine, a natural alkaloid derived from a medicinal plant species, Berberis which has been shown to possess anti-inflammatory and anti-cancer properties with no known toxicity; however, the effect of berberine against HPV has not been elucidated.
We studied the effect of berberine on HPV16-positive cervical cancer cell line, SiHa and HPV18-positive cervical cancer cell line, HeLa using electrophoretic mobility gel shift assays, western and northern blotting which showed that berberine could selectively inhibit constitutively activated AP-1 in a dose- and time-dependent manner and downregulates HPV oncogenes expression. Inhibition of AP-1 was also accompanied by changes in the composition of their DNA-binding complex. Berberine specifically downregulated expression of oncogenic c-Fos which was also absent in the AP-1 binding complex. Treatment with berberine resulted in repression of E6 and E7 levels and concomitant increase in p53 and Rb expression in both cell types. Berberine also suppressed expression of telomerase protein, hTERT, which translated into growth inhibition of cervical cancer cells. Interestingly, a higher concentration of berberine was found to reduce the cell viability through mitochondria-mediated pathway and induce apoptosis by activating caspase-3.
These results indicate that berberine can effectively target both the host and viral factors responsible for development of cervical cancer through inhibition of AP-1 and blocking viral oncoproteins E6 and E7 expression. Inhibition of AP-1 activity by berberine may be one of the mechanisms responsible for the anti-HPV effect of berberine. We propose that berberine is a potentially promising compound for the treatment of cervical cancer infected with HPV.
Cervical cancer is one of the most common cancers in women. More than 275,100 women die from cervical cancer each year. Cervical squamous cell carcinoma (cervical SCC), one of the most frequent types of cervical cancers, is associated with high-risk human papilloma virus (HPV), although HPV infection alone may not be enough to induce malignant transformation. MicroRNAs (miRNAs), a class of small non-coding RNAs, regulate protein-coding gene expression by repressing translation or cleaving RNA transcripts in a sequence-specific manner. A growing body of evidence suggests that miRNAs contribute to cervical SCC progression, development and metastasis. miRNA expression signatures in SCC (hypopharyngeal SCC and esophageal SCC) revealed that miR-218 expression was significantly reduced in cancer tissues compared with adjacent non-cancerous epithelium, suggesting that miR-218 is a candidate tumor suppressor. The aim of this study was to investigate the functional significance of miR-218 in cervical SCC and to identify novel miR-218-mediated cancer pathways in cervical SCC. Restoration of miR-218 significantly inhibited cancer cell migration and invasion in both HPV-positive and HPV-negative cervical SCC cell lines. These data indicated that miR-218 acts as a tumor suppressor in cervical SCC. Our in silico analysis showed that miR-218 appeared to be an important modulator of tumor cell processes through suppression of many targets, particularly those involved in focal adhesion signaling pathways. Gene expression data indicated that LAMB3, a laminin protein known to influence cell differentiation, migration, adhesion, proliferation and survival, was upregulated in cervical SCC clinical specimens, and silencing studies demonstrated that LAMB3 functioned as an oncogene in cervical SCC. The identification of novel tumor-suppressive miR-218-mediated molecular pathways has provided new insights into cervical SCC oncogenesis and metastasis.
microRNA; miR-218; tumor suppressor; cervical cancer; LAMB3; focal adhesion
Infection by high-risk human papillomaviruses (HPV) and persistent expression of viral oncogenes E6 and E7 are causally linked to the development of cervical cancer. These oncogenes are necessary but insufficient for complete transformation of human epithelial cells in vivo. Intracellular Notch1 protein is detected in invasive cervical carcinomas (ICC), and truncated Notch1 alleles complement the function of E6/E7 in the transformation of human epithelial cells. Here we investigate potential mechanisms of Notch activation in a human cervical neoplasia. We have analyzed human cervical lesions and serial passages of an HPV type 16-positive human cervical low-grade lesion-derived cell line, W12, that shows abnormalities resembling those seen in cervical neoplastic progression in vivo. Late-passage, but not early-passage, W12 and progression of the majority of human high-grade cervical lesions to ICC showed upregulation of Notch ligand and Jagged1 and downregulation of Manic Fringe, a negative regulator of Jagged1-Notch1 signaling. Concomitantly, an increase in Notch/CSL (CBF1, Suppressor of Hairless, Lag1)-driven reporter activity and a decrease in Manic Fringe upstream regulatory region (MFng-URR)-driven reporter activity was observed in late-passage versus early passage W12. Analysis of the MFng-URR revealed that Notch signaling represses this gene through Hairy Enhancer of Split 1, a transcriptional target of the Notch pathway. Expression of Manic Fringe by a recombinant adenovirus, dominant-negative Jagged1, or small interfering RNA against Jagged1 inhibits the tumorigenicity of CaSki, an ICC-derived cell line that was previously shown to be susceptible to growth inhibition induced by antisense Notch1. We suggest that activation of Notch in cervical neoplasia is Jagged1 dependent and that its susceptibility to the influence of Manic Fringe is of therapeutic value.
High-risk human papillomavirus (HPV) must evade innate immune surveillance to establish persistent infections and to amplify viral genomes upon differentiation. Members of the JAK-STAT family are important regulators of the innate immune response and HPV proteins downregulate expression of STAT-1 to allow for stable maintenance of viral episomes. STAT-5 is another member of this pathway that modulates the inflammatory response and plays an important role in controlling cell cycle progression in response to cytokines and growth factors. Our studies show that HPV E7 activates STAT-5 phosphorylation without altering total protein levels. Inhibition of STAT-5 phosphorylation by the drug pimozide abolishes viral genome amplification and late gene expression in differentiating keratinocytes. In contrast, treatment of undifferentiated cells that stably maintain episomes has no effect on viral replication. Knockdown studies show that the STAT-5β isoform is mainly responsible for this activity and that this is mediated through the ATM DNA damage response. A downstream target of STAT-5, the peroxisome proliferator-activated receptor γ (PPARγ) contributes to the effects on members of the ATM pathway. Overall, these findings identify an important new regulatory mechanism by which the innate immune regulator, STAT-5, promotes HPV viral replication through activation of the ATM DNA damage response.
Over 120 types of human papillomavirus (HPV) have been identified, and approximately one-third of these infect epithelial cells of the genital mucosa. A subset of HPV types are the causative agents of cervical and other anogenital cancers. The infectious life cycle of HPV is dependent on differentiation of the host epithelial cell, with viral genome amplification and virion production restricted to differentiated suprabasal cells. While normal keratinocytes exit the cell cycle upon differentiation, HPV-positive suprabasal cells are able to re-enter S-phase to mediate productive replication. HPV induces an ATM-dependent DNA damage response in differentiating cells that is essential for viral genome amplification. Our studies describe an important mechanism by which human papillomaviruses activate a member of the JAK/STAT innate immune signaling pathway to induce the ATM DNA damage pathway. This is necessary for differentiation-dependent productive viral replication. HPVs must suppress the transcription of one member of the JAK/STAT pathway, STAT-1, while at the same time activating STAT-5 to regulate genome amplification in suprabasal cells. The E7 protein activates STAT-5 leading to induction of ATM phosphorylation through the PPARγ pathway. Our study identifies important links between innate immune signaling, the ATM DNA damage pathway and productive HPV replication that may lead to the characterization of new targets for the development of therapeutics to treat HPV-induced infections.
To discover cancer specific DNA methylation markers, large-scale screening methods are widely used. The pharmacological unmasking expression microarray approach is an elegant method to enrich for genes that are silenced and re-expressed during functional reversal of DNA methylation upon treatment with demethylation agents. However, such experiments are performed in in vitro (cancer) cell lines, mostly with poor relevance when extrapolating to primary cancers. To overcome this problem, we incorporated data from primary cancer samples in the experimental design. A strategy to combine and rank data from these different data sources is essential to minimize the experimental work in the validation steps.
To apply a new relaxation ranking algorithm to enrich DNA methylation markers in cervical cancer.
The application of a new sorting methodology allowed us to sort high-throughput microarray data from both cervical cancer cell lines and primary cervical cancer samples. The performance of the sorting was analyzed in silico. Pathway and gene ontology analysis was performed on the top-selection and gives a strong indication that the ranking methodology is able to enrich towards genes that might be methylated. Terms like regulation of progression through cell cycle, positive regulation of programmed cell death as well as organ development and embryonic development are overrepresented. Combined with the highly enriched number of imprinted and X-chromosome located genes, and increased prevalence of known methylation markers selected from cervical (the highest-ranking known gene is CCNA1) as well as from other cancer types, the use of the ranking algorithm seems to be powerful in enriching towards methylated genes.
Verification of the DNA methylation state of the 10 highest-ranking genes revealed that 7/9 (78%) gene promoters showed DNA methylation in cervical carcinomas. Of these 7 genes, 3 (SST, HTRA3 and NPTX1) are not methylated in normal cervix tissue.
The application of this new relaxation ranking methodology allowed us to significantly enrich towards methylation genes in cancer. This enrichment is both shown in silico and by experimental validation, and revealed novel methylation markers as proof-of-concept that might be useful in early cancer detection in cervical scrapings.
Human papilloma virus (HPV) infection of the uterine cervix is linked to the pathogenesis of cervical cancer. Preclinical in vitro and in vivo studies using HPV-containing human cervical carcinoma cell lines have shown that the mammalian target of rapamycin (mTOR) inhibitor, rapamycin, and epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor, erlotinib, can induce growth delay of xenografts. Activation of Akt and mTOR are also observed in cervical squamous cell carcinoma and, the expression of phosphorylated mTOR was reported to serve as a marker to predict response to chemotherapy and survival of cervical cancer patients. Therefore, we investigated: a) the expression level of EGFR in cervical squamous cell carcinoma (SCC) and high-grade squamous intraepithelial lesions (HSIL) versus non-neoplastic cervical squamous epithelium; b) the state of activation of the mTOR pathway in these same tissues; and c) any impact of these signal transduction molecules on cell cycle. Formalin-fixed paraffin-embedded tissue microarray blocks containing 20 samples each of normal cervix, HSIL and invasive SCC, derived from a total of 60 cases of cervical biopsies and cervical conizations were examined. Immunohistochemistry was utilized to detect the following antigens: EGFR; mTOR pathway markers, phosphorylated (p)-mTOR (Ser2448) and p-p70S6K (Thr389); and cell cycle associated proteins, Ki-67 and S phase kinase-associated protein (Skp)2. Protein compartmentalization and expression were quantified in regard to proportion (0-100%) and intensity (0-3+). Mitotic index (MI) was also assessed. An expression index (EI) for pmTOR, p-p70S6K and EGFR, respectively was calculated by taking the product of intensity score and proportion of positively staining cells. We found that plasmalemmal EGFR expression was limited to the basal/parabasal cells (2-3+, EI = 67) in normal cervical epithelium (NL), but was diffusely positive in all HSIL (EI = 237) and SCC (EI 226). The pattern of cytoplasmic p-mTOR and nuclear p-p70S6K expression was similar to that of EGFR; all showed a significantly increased EI in HSIL/SCC versus NL (p<0.02). Nuclear translocation of p-mTOR was observed in all SCC lesions (EI = 202) and was significantly increased versus both HSIL (EI = 89) and NL (EI = 54) with p<0.015 and p<0.0001, respectively. Concomitant increases in MI and proportion of nuclear Ki-67 and Skp2 expression were noted in HSIL and SCC. In conclusion, morphoproteomic analysis reveals constitutive activation and overexpression of the mTOR pathway in HSIL and SCC as evidenced by: increased nuclear translocation of pmTOR and p-p70S6K, phosphorylated at putative sites of activation, Ser2448 and Thr389, respectively; correlative overexpression of the upstream signal transducer, EGFR, and increases in cell cycle correlates, Skp2 and mitotic indices. These results suggest that the mTOR pathway plays a key role in cervical carcinogenesis and targeted therapies may be developed for SCC as well as its precursor lesion, HSIL.
mTOR pathway; cervical squamous carcinoma; high grade SIL; morphoproteomics
Somatic mutations in the p53 tumor suppressor gene represent the single most common genetic alteration observed in human cancers. Interestingly, the great majority of malignant tumors of the cervix uteri contain wild-type p53 alleles together with the DNA of specific types of human papillomaviruses (HPVs), while the small portion of HPV-negative cervical carcinomas often carry alterations in the p53 tumor suppressor gene. Transcriptional activation of yet-undefined cellular regulatory genes has been implicated to play a key role for the tumor-suppressive activity of wild-type p53, as mutant p53 in general has lost the activity to stimulate p53-responsive reporter plasmids. The detection of DNA-binding-competent and transcriptionally active p53 protein in HeLa cervical carcinoma cells enabled us to investigate the in vivo effects of putative modulators on endogenous p53 function in cervical cancer cells. We show that the transcriptional stimulatory activity of HeLa cell p53 is strongly repressed by overexpression of E6 protein from oncogenic HPV type 16 (HPV16) but is not influenced by low-risk HPV6 E6. Similar to HPV16 E6, cellular oncoproteins such as mutant p53 or the product of the human mdm-2 gene also negatively interfere with p53-mediated transactivation in HeLa cells. Our findings indicate that, within a cervical cancer cell, the expression of E6 protein from high-risk HPV16, but not from low-risk HPV6, can lead to the same functional consequences as a mutation of the p53 gene. These results could provide a biochemical basis for the inverse correlation between the presence of HPV sequences and somatic mutations of the p53 gene in cervical carcinomas.
Infection with genital human papillomaviruses (HPVs) is the primary cause of cervical cancer. The infection is widespread, and little is known about the secondary factors associated with progression from subclinical infection to invasive carcinoma. Here we report that HPV genomes are efficiently targeted in vivo by CpG methylation, a well-known mechanism of transcriptional repression. Indeed, it has been shown previously that in vitro-methylated HPV type 16 (HPV-16) DNA is transcriptionally repressed after transfection into cell cultures. By using a scan with the restriction enzyme McrBC, we observed a conserved profile of CpG hyper- and hypomethylation throughout the HPV-16 genomes of the tumor-derived cell lines SiHa and CaSki. Methylation is particularly high in genomic segments overlying the late genes, while the long control region (LCR) and the oncogenes are unmethylated in the single HPV-16 copy in SiHa cells. In 81 patients from two different cohorts, the LCR and the E6 gene of HPV-16 DNA were found to be hypermethylated in 52% of asymptomatic smears, 21.7% of precursor lesions, and 6.1% of invasive carcinomas. This suggests that neoplastic transformation may be suppressed by CpG methylation, while demethylation occurs as the cause of or concomitant with neoplastic progression. These prevalences of hyper- and hypomethylation also indicate that CpG methylation plays an important role in the papillomavirus life cycle, which takes place in asymptomatic infections and precursor lesions but not in carcinomas. Bisulfite modification revealed that in most of the HPV-16 genomes of CaSki cells and of asymptomatic patients, all 11 CpG dinucleotides that overlap with the enhancer and the promoter were methylated, while in SiHa cells and cervical lesions, the same 11 or a subset of CpGs remained unmethylated. Our report introduces papillomaviruses as models to study the mechanism of CpG methylation, opens research on the importance of this mechanism during the viral life cycle, and provides a marker relevant for the etiology and diagnosis of cervical cancer.
The expression of the human papillomavirus (HPV) E6/E7 oncogenes is crucial for HPV-induced malignant cell transformation. The identification of cellular targets attacked by the HPV oncogenes is critical for our understanding of the molecular mechanisms of HPV-associated carcinogenesis and may open novel therapeutic opportunities. Here, we identify the Lens Epithelial-Derived Growth Factor (LEDGF) gene as a novel cellular target gene for the HPV oncogenes. Elevated LEDGF expression has been recently linked to human carcinogenesis and can protect tumor cells towards different forms of cellular stress. We show that intracellular LEDGF mRNA and protein levels in HPV-positive cancer cells are critically dependent on the maintenance of viral oncogene expression. Ectopic E6/E7 expression stimulates LEDGF transcription in primary keratinocytes, at least in part via activation of the LEDGF promoter. Repression of endogenous LEDGF expression by RNA interference results in an increased sensitivity of HPV-positive cancer cells towards genotoxic agents. Immunohistochemical analyses of cervical tissue specimens reveal a highly significant increase of LEDGF protein levels in HPV-positive lesions compared to histologically normal cervical epithelium. Taken together, these results indicate that the E6/E7-dependent maintenance of intracellular LEDGF expression is critical for protecting HPV-positive cancer cells against various forms of cellular stress, including DNA damage. This could support tumor cell survival and contribute to the therapeutic resistance of cervical cancers towards genotoxic treatment strategies in the clinic.
Specific types of human papillomaviruses (HPVs) are closely linked to the development of malignant tumors, such as cervical cancer. Virtually all cervical cancers contain HPV DNA and the tumorigenic growth behavior of cervical cancer cells is dependent on the activity of two viral oncogenes, called E6 and E7. It is important to study the activities by which the HPV oncogenes can support the growth of tumor cells. This should allow new insights into the molecular mechanisms of virus-induced carcinogenesis and could also be useful for developing novel approaches for cancer therapy. We here show that the HPV oncogenes stimulate and maintain expression of the cellular LEDGF gene in HPV-positive cancer cells. Consistently, pre-malignant and malignant lesions of the cervix exhibit significantly increased LEDGF protein levels. LEDGF is crucial for the protection of tumor cells against various forms of cellular stress, including DNA damage. LEDGF stimulation by the viral oncogenes could be a critical survival mechanism by which HPVs support the growth of cervical cancer cells and provide resistance towards chemo- and radiotherapy in the clinic.
Hepatitis C virus (HCV) is a positive-strand RNA virus that frequently causes persistent infections and is uniquely associated with the development of hepatocellular carcinoma. While the mechanism(s) by which the virus promotes cancer are poorly defined, previous studies indicate that the HCV RNA-dependent RNA polymerase, nonstructural protein 5B (NS5B), forms a complex with the retinoblastoma tumor suppressor protein (pRb), targeting it for degradation, activating E2F-responsive promoters, and stimulating cellular proliferation. Here, we describe the mechanism underlying pRb regulation by HCV and its relevance to HCV infection. We show that the abundance of pRb is strongly downregulated, and its normal nuclear localization altered to include a major cytoplasmic component, following infection of cultured hepatoma cells with either genotype 1a or 2a HCV. We further demonstrate that this is due to NS5B-dependent ubiquitination of pRb and its subsequent degradation via the proteasome. The NS5B-dependent ubiquitination of pRb requires the ubiquitin ligase activity of E6-associated protein (E6AP), as pRb abundance was restored by siRNA knockdown of E6AP or overexpression of a dominant-negative E6AP mutant in cells containing HCV RNA replicons. E6AP also forms a complex with pRb in an NS5B-dependent manner. These findings suggest a novel mechanism for the regulation of pRb in which the HCV NS5B protein traps pRb in the cytoplasm, and subsequently recruits E6AP to this complex in a process that leads to the ubiquitination of pRb. The disruption of pRb/E2F regulatory pathways in cells infected with HCV is likely to promote hepatocellular proliferation and chromosomal instability, factors important for the development of liver cancer.
Persons infected with hepatitis C virus (HCV) are at increased risk for liver cancer. This is remarkable because HCV is an RNA virus with replication confined to the cytoplasm and no potential for integration of its genome into host cell DNA. While it is likely that chronic inflammation contributes to liver cancer, prior studies with HCV transgenic mice indicate that the viral proteins are intrinsically carcinogenic. In this study, we have examined the interaction of one of these, the RNA-dependent RNA polymerase nonstructural protein 5B, with an important cellular tumor suppressor protein, the retinoblastoma protein (pRb). pRb is a master regulator of the cell cycle, and altered expression of some of the many genes it regulates may lead to cancer. We show that the abundance of pRb is strongly downregulated in cells infected with HCV, and that nonstructural protein 5B targets pRb for destruction via the cell's normal protein degradation machinery. The E6-associated protein appears to play a role in this process, which is interesting as it also mediates the degradation of another tumor suppressor, p53, by papillomaviruses. The loss of pRb function in HCV-infected cells likely promotes hepatocellular proliferation as well chromosomal instability, factors important for the development of liver cancer.
Human papillomaviruses (HPVs) are the etiological agents for genital warts and contribute to the development of cervical cancer in humans. The HPV E7 gene product is expressed in these diseases, and the E7 genes from HPV types 16 and 18 contribute to transformation in mammalian cells. Mutation and deletion analysis of this gene suggests that the transforming activity of the protein product resides in the same domain as that which is directly involved in complex formation with the retinoblastoma gene product (pRB). This domain is one of two conserved regions (designated CRI and CRII) shared by E7 and other viral oncoproteins which bind pRB, including adenovirus E1A protein. Binding of HPV type 16 E7 protein to pRB has previously been shown to affect pRB's ability to bind DNA and to form complexes with other cellular proteins. In the current study, we map the functional interaction between E7 protein and pRB by monitoring the association between a 60-kDa version of the pRB, pRB60, and the cellular transcription factor E2F. We observe that CRII of E7 (amino acids 20 to 29), which completely blocks binding of full-length E7 protein, is necessary but not sufficient to inhibit E2F/pRB60 complex formation. While CRI of E1A (amino acids 37 to 55) appears to be sufficient to compete with E2F for binding to pRB60, the equivalent region of E7 is neither necessary nor sufficient. Only E7 fragments that contained both CRII and at least a portion of the zinc-binding domain (amino acids 60 to 98) inhibited E2F/pRB60 complex formation. These results suggest that pRB60 associates with E7 and E2F through overlapping but distinct domains.
The transforming genes E6 and E7 of high-risk human papillomaviruses are consistently expressed in papillomavirus-associated neoplasms of the anogenital tract. In papillomavirus type 18-associated SW 756 cervical carcinoma cells, transcription of the viral E6-E7 genes is blocked by dexamethasone. Herein we show that dexamethasone-mediated repression of the E6-E7 genes results in loss of the neoplastic phenotype of SW 756 cells. Withdrawal of dexamethasone restores E6-E7 expression and neoplastic growth. Moreover, reconstitution of E6-E7 gene expression by a dexamethasone-inducible expression vector renders the neoplastic phenotype resistant to dexamethasone. These results clearly indicate that the continuous expression of the viral E6-E7 oncogenes is required to maintain the neoplastic growth properties of SW 756 cervical cancer cells. The viral E6 protein destabilizes the p53 tumor suppressor gene product in vitro. Since low levels of p53 have been observed in papillomavirus-transformed keratinocyte cell lines, it was speculated that degradation of p53 by E6 contributes to papillomavirus-associated growth deregulation. Consistent with this hypothesis, we detected a significant increase in p53 levels upon dexamethasone-induced repression of papillomavirus E6-E7 oncogene expression. No p53 increase was observed in dexamethasone-treated cells in which the viral oncogene expression was restored. The viral E7 protein has been shown to complex with the retinoblastoma tumor suppressor gene product (pRB). In some cells, this interaction has been shown shown to release the transcription factor E2F from its complex with pRB, and it has been hypothesized that E7-induced, increased levels of free E2F contribute to the transforming potential of the viral oncogenes. In gel shift experiments, we detected relatively stable complexes of pRB and E2F in all SW 756-derived cells, independent of the level of E7 expression. This suggests that E7-mediated release of E2F from its complex with pRB might not be required to maintain the neoplastic phenotype of human papillomavirus-associated cancer cells, although a possibly relevant partial E7-mediated release of E2F from pRB cannot be excluded.
Apoptotic cell death is an important response to genotoxic stress that prevents oncogenesis. It is known that tissues can differ in their apoptotic response, but molecular mechanisms are little understood. Here, we show that Drosophila polyploid endocycling cells (G/S cycle) repress the apoptotic response to DNA damage through at least two mechanisms. First, the expression of all the Drosophila p53 protein isoforms is strongly repressed at a post-transcriptional step. Second, p53-regulated pro-apoptotic genes are epigenetically silenced in endocycling cells, preventing activation of a paused RNA Pol II by p53-dependent or p53-independent pathways. Over-expression of the p53A isoform did not activate this paused RNA Pol II complex in endocycling cells, but over-expression of the p53B isoform with a longer transactivation domain did, suggesting that dampened p53B protein levels are crucial for apoptotic repression. We also find that the p53A protein isoform is ubiquitinated and degraded by the proteasome in endocycling cells. In mitotic cycling cells, p53A was the only isoform expressed to detectable levels, and its mRNA and protein levels increased after irradiation, but there was no evidence for an increase in protein stability. However, our data suggest that p53A protein stability is regulated in unirradiated cells, which likely ensures that apoptosis does not occur in the absence of stress. Without irradiation, both p53A protein and a paused RNA pol II were pre-bound to the promoters of pro-apoptotic genes, preparing mitotic cycling cells for a rapid apoptotic response to genotoxic stress. Together, our results define molecular mechanisms by which different cells in development modulate their apoptotic response, with broader significance for the survival of normal and cancer polyploid cells in mammals.
In order to maintain genome integrity, eukaryotic cells have evolved multiple ways to respond to DNA damage stress. One of the major cellular responses is apoptosis, during which the cell undergoes programmed cell death in order to prevent the propagation of the damaged genome to daughter cells. Although clinical observations and other studies have shown that tissues can differ in their apoptotic response, the molecular mechanisms underlying these differences are largely unknown. We have shown in our model system, Drosophila, that endocycling cells do not initiate cell death in response to DNA damage. The endocycle is a cell cycle variation that is widely found in nature and conserved from plant to animals. During the endocycle, cells duplicate their genomic DNA but do not enter mitosis to segregate chromosomes, resulting in a polyploid genome content. In this study, we investigate how the apoptotic response to DNA damage is repressed in endocycling cells. We find that the Drosophila ortholog of the human p53 tumor suppressor protein is expressed at very low levels in endocycling cells. Moreover, the downstream pro-apoptotic genes that are regulated by p53 are epigenetically silenced in endocycling cells. Our results provide important insights into tissue-specific apoptotic responses in development, with possible broader impact on understanding radiation therapy response and cancer of different tissues.
High-risk human papillomavirus oncoproteins E6 and E7 play a major role in HPV-related cancers. One of the main functions of E7 is the degradation of pRb, while E6 promotes the degradation of p53, inactivating the p14ARF-p53 pathway. pRb and p14ARF can repress ribosomal DNA (rDNA) transcription in part by targeting the Upstream Binding Factor 1 (UBF1), a key factor in the activation of RNA polymerase I machinery. We showed, through ectopic expression and siRNA silencing of p14ARF and/or E7, that E7 stimulates UBF1-mediated rDNA gene transcription, partly because of increased levels of phosphorylated UBF1, preventing the inhibitory function of p14ARF. Unexpectedly, activation of rDNA gene transcription was higher in cells co-expressing p14ARF and E7, compared to cells expressing E7 alone. We did not find a difference in P-UBF1 levels that could explain this data. However, p14ARF expression induced E7 to accumulate into the nucleolus, where rDNA transcription takes place, providing an opportunity for E7 to interact with nucleolar proteins involved in this process. GST-pull down and co-immunoprecipitation assays showed interactions between p14ARF, UBF1 and E7, although p14ARF and E7 are not able to directly interact. Co-expression of a pRb-binding-deficient mutant (E7C24G) and p14ARF resulted in EC24G nucleolar accumulation, but not in a significant higher activation of rDNA transcription, suggesting that the inactivation of pRb is involved in this phenomenon. Thus, p14ARF fails to prevent E7-mediated UBF1 phosphorylation, but could facilitate nucleolar pRb inactivation by targeting E7 to the nucleolus. While others have reported that p19ARF, the mouse homologue of p14ARF, inhibits some functions of E7, we showed that E7 inhibits a p53-independent function of p14ARF. These results point to a mutually functional interaction between p14ARF and E7 that might partly explain why the sustained p14ARF expression observed in most cervical pre-malignant lesions and malignancies may be ineffective.
Expression of the high-risk human papillomavirus (HPV) E6 and E7 oncogenes is essential for the initiation and maintenance of cervical cancer. The repression of both was previously shown to result in activation of their respective tumor suppressor targets, p53 and pRb, and subsequent senescence induction in cervical cancer cells. Consequently, viral oncogene suppression is a promising approach for the treatment of HPV-positive tumors. One well-established method of E6/E7 repression involves the reexpression of the viral E2 protein which is usually deleted in HPV-positive cancer cells. Here, we show that, surprisingly, bovine papillomavirus type 1 (BPV1) E2 but not RNA interference-mediated E6/E7 repression in HPV-positive cervical cancer cells stimulates cellular motility and invasion. Migration correlated with the dynamic formation of cellular protrusions and was dependent upon cell-to-cell contact. While E2-expressing migratory cells were senescent, migration was not a general feature of cellular senescence or cell cycle arrest and was specifically observed in HPV-positive cervical cancer cells. Interestingly, E2-expressing cells not only were themselves motile but also conferred increased motility to admixed HeLa cervical cancer cells. Together, our data suggest that repression of the viral oncogenes by E2 stimulates the motility of E6/E7-targeted cells as well as adjacent nontargeted cancer cells, thus raising the possibility that E2 expression may unfavorably increase the local invasiveness of HPV-positive tumors.
Carcinoma of the uterine cervix is one of the most common malignancies among women worldwide. Human papillomaviruses (HPV) have been identified as the major etiological factor in cervical carcinogenesis. However, the time lag between HPV infection and the diagnosis of cancer indicates that multiple steps, as well as multiple factors, may be necessary for the development of cervical cancer. The development and progression of cervical carcinoma have been shown to be dependent on various genetic and epigenetic events, especially alterations in the cell cycle checkpoint machinery. In mammalian cells, control of the cell cycle is regulated by the activity of cyclin-dependent kinases (CDKs) and their essential activating coenzymes, the cyclins. Generally, CDKs, cyclins, and CDK inhibitors function within several pathways, including the p16INK4A-cyclin D1-CDK4/6-pRb-E2F, p21WAF1-p27KIP1-cyclinE-CDK2, and p14ARF-MDM2-p53 pathways. The results from several studies showed aberrant regulation of several cell cycle proteins, such as cyclin D, cyclin E, p16INK4A, p21WAF1, and p27KIP1, as characteristic features of HPV-infected and HPV E6/E7 oncogene-expressing cervical carcinomas and their precursors. These data suggested further that interactions of viral proteins with host cellular proteins, particularly cell cycle proteins, are involved in the activation or repression of cell cycle progression in cervical carcinogenesis.
Cell cycle; cervical carcinoma; cyclin
Nucleoporins mediate nucleocytoplasmic exchange of macromolecules and several have been assigned active mitotic functions. Nucleoporins can participate in various mitotic functions like spindle assembly, kinetochore organisation and chromosome segregation- important for genome integrity. Pathways to genome integrity are frequently deregulated in cancer and many are regulated in part by microRNAs. Indeed, altered levels of numerous microRNAs have frequently been associated with tumorigenesis. Here, we unveil a microRNA-mediated regulation of the nucleoporin Nup214 and its downstream effect on genome integrity.
Databases/bioinformatic tools such as miRBase, Oncomine and RNAhybrid predicted Nup214 as a miR-133b target. To validate this, we used luciferase reporter assays, Real-Time PCR and immuno-blotting. Flow cytometry and immuno-blots of mitotic markers were used to analyse cell cycle pattern upon thymidine synchronization and miR-133b treatment. Mitotic indices and chromosomal abnormalities were assessed by immuno-fluorescence for FITC-tagged phospho-H3 as well as video-microscopy for GFP-tagged histone H4. Annexin V/propidium iodide staining, caspase3/PARP cleavage and colony formation assays were done to investigate cell death upon either miR-133b transfection or NUP214 knockdown by siRNA. UPCI:SCC084, HCT116, HeLa-H4-pEGFP and HEK293 (human oral squamous cell carcinoma, colorectal, cervical carcinomas and embryonic kidney cell lines, respectively) were used. miR-133b and NUP214 expressions were validated in cancer cell lines and tissues by Real-Time PCR.
Examination of head and neck tumour tissues and cancer cell lines revealed that Nup214 and miR-133b expressions are negatively correlated. In vitro, Nup214 was significantly downregulated by ectopic miR-133b. This downregulation elevated mitotic indices and delayed degradation of mitotic marker proteins cyclinB1 and cyclinA and dephosphorylation of H3. Moreover, this mitotic delay enhanced chromosomal abnormalities and apoptosis.
We have identified NUP214, a member of the massive nuclear pore complex, as a novel miR-133b target. Thus, we have shown a hitherto unknown microRNA regulation of mitosis mediated by a member of the nucleoporin family. Based on observations, we also raise some hypotheses regarding transport-dependent/independent functions of Nup214 in this study. Our results hence attempt to explain why miR-133b is generally downregulated in tumours and lay out the potential for Nup214 as a therapeutic target in the treatment of cancer.
Electronic supplementary material
The online version of this article (doi:10.1186/s12943-015-0299-z) contains supplementary material, which is available to authorized users.
Nucleoporin; Nup214; miR-133b; Mitosis; Apoptosis; Cell cycle; MicroRNA; Cancer; Head and neck cancer; Chromosomal abnormality
Specific types of human papillomaviruses (HPVs) cause cervical cancer. Cervical cancers exhibit aberrant cellular microRNA (miRNA) expression patterns. By genome-wide analyses, we investigate whether the intracellular and exosomal miRNA compositions of HPV-positive cancer cells are dependent on endogenous E6/E7 oncogene expression. Deep sequencing studies combined with qRT-PCR analyses show that E6/E7 silencing significantly affects ten of the 52 most abundant intracellular miRNAs in HPV18-positive HeLa cells, downregulating miR-17-5p, miR-186-5p, miR-378a-3p, miR-378f, miR-629-5p and miR-7-5p, and upregulating miR-143-3p, miR-23a-3p, miR-23b-3p and miR-27b-3p. The effects of E6/E7 silencing on miRNA levels are mainly not dependent on p53 and similarly observed in HPV16-positive SiHa cells. The E6/E7-regulated miRNAs are enriched for species involved in the control of cell proliferation, senescence and apoptosis, suggesting that they contribute to the growth of HPV-positive cancer cells. Consistently, we show that sustained E6/E7 expression is required to maintain the intracellular levels of members of the miR-17~92 cluster, which reduce expression of the anti-proliferative p21 gene in HPV-positive cancer cells. In exosomes secreted by HeLa cells, a distinct seven-miRNA-signature was identified among the most abundant miRNAs, with significant downregulation of let-7d-5p, miR-20a-5p, miR-378a-3p, miR-423-3p, miR-7-5p, miR-92a-3p and upregulation of miR-21-5p, upon E6/E7 silencing. Several of the E6/E7-dependent exosomal miRNAs have also been linked to the control of cell proliferation and apoptosis. This study represents the first global analysis of intracellular and exosomal miRNAs and shows that viral oncogene expression affects the abundance of multiple miRNAs likely contributing to the E6/E7-dependent growth of HPV-positive cancer cells.
Oncogenic human papillomaviruses (HPVs) are major human carcinogens of broad biomedical importance. The growth of HPV-positive cervical cancer cells is critically dependent on sustained E6/E7 oncogene expression from endogenous viral DNA sequences. We here addressed the question of whether this process is linked to specific, E6/E7-dependent alterations of the cellular micro(mi)RNA network. By comprehensive deep sequencing analyses we show that endogenous E6/E7 expression significantly affects the concentrations of abundant intracellular miRNAs in HPV-positive cervical cancer cells, which are linked to the control of cell proliferation, senescence and apoptosis. These include members of the miR-17~92 cluster, which are expressed at increased levels by sustained E6/E7 expression and repress the anti-proliferative p21 gene in HPV-positive cancer cells. Moreover, we identified an E6/E7-dependent seven-miRNA-signature in exosomes secreted from HPV-positive cancer cells. These small vesicles are involved in intercellular communication and may serve as novel diagnostic markers. Taken together, our results show that continuous E6/E7 expression in HPV-positive cancer cells is linked to significant alterations in the amounts of intracellular and exosomal miRNAs with growth-promoting, anti-senescent and anti-apoptotic potential.
Aberrant activation of the Wnt/β-catenin signaling axis is a prominent oncogenic mechanism in numerous cancers including cervical cancer. Wnt inhibitory factor-1 (WIF1) is a secreted protein that binds Wnt and antagonizes Wnt activity. While the WIF1 gene is characterized as a target for epigenetic silencing in some tumor types, WIF1 expression has not been examined in human cervical tissue and cervical cancer. Here, we show that WIF1 is unmethylated and its gene product is expressed in normal cervical epithelium and some cultured cervical tumor lines. In contrast, several cervical cancer lines contained dense CpG methylation within the WIF1 gene, and expression of both WIF1 transcript and protein was restored by culturing cells in the presence of the global DNA demethylating agent 5-aza-2′-deoxycytidine. Using single-molecule MAPit methylation footprinting, we observed differences in chromatin structure within the WIF1 promoter region between cell lines that express and those that do not express WIF1, consistent with transcriptional activity and repression, respectively. The WIF1 promoter was aberrantly methylated in ∼60% (10 of 17) high-grade highly undifferentiated squamous cell cervical tumors examined, whereas paired normal tissue showed significantly lower levels of CpG methylation. WIF1 protein was not detectable by immunohistochemistry in tumors with quantitatively high levels of WIF1 methylation. Of note, WIF1 protein was not detectable in two of the seven unmethylated cervical tumors examined, suggesting other mechanisms may contribute WIF1 repression. Our findings establish the WIF1 gene as a frequent target for epigenetic silencing in squamous cell carcinoma of the cervix.
Mechanisms that maintain transcriptional memory through cell division are important to maintain cell identity, and sequence-specific transcription factors that remain associated with mitotic chromatin are emerging as key players in transcriptional memory propagation. Here, we show that the major transcriptional effector of Notch signaling, RBPJ, is retained on mitotic chromatin, and that this mitotic chromatin association is mediated through the direct association of RBPJ with DNA. We further demonstrate that RBPJ binds directly to nucleosomal DNA in vitro, with a preference for sites close to the entry/exit position of the nucleosomal DNA. Genome-wide analysis in the murine embryonal-carcinoma cell line F9 revealed that roughly 60% of the sites occupied by RBPJ in asynchronous cells were also occupied in mitotic cells. Among them, we found that a fraction of RBPJ occupancy sites shifted between interphase and mitosis, suggesting that RBPJ can be retained on mitotic chromatin by sliding on DNA rather than disengaging from chromatin during mitotic chromatin condensation. We propose that RBPJ can function as a mitotic bookmark, marking genes for efficient transcriptional activation or repression upon mitotic exit. Strikingly, we found that sites of RBPJ occupancy were enriched for CTCF-binding motifs in addition to RBPJ-binding motifs, and that RBPJ and CTCF interact. Given that CTCF regulates transcription and bridges long-range chromatin interactions, our results raise the intriguing hypothesis that by collaborating with CTCF, RBPJ may participate in establishing chromatin domains and/or long-range chromatin interactions that could be propagated through cell division to maintain gene expression programs.
How does a cell remember what it should be after cell division? One mechanism that is beginning to emerge is the retention of a few key regulatory proteins on the highly condensed mitotic chromatin during cell division. These proteins are called mitotic bookmarks, as they are believed to offer critical information as to how genetic information should be read immediately after mitosis. We have found that a protein called RBPJ, which plays pivotal roles in regulating cell-fate choices, is retained on mitotic chromatin. RBPJ transmits to DNA signals elicited by the Notch pathway: a pathway that conveys information resulting from the communication between two adjacent cells. Unlike many other factors, we found that RBPJ can bind to nucleosomes, which are the basic unit of packaged DNA consisting of DNA wrapped around eight histone proteins. We also found that RBPJ interacts with and binds to DNA sites regulated by the CTCF protein, which plays important roles in regulating long-range DNA interactions. Together, our results suggest that RBPJ can function as a mitotic bookmarking factor, to help maintain genetic programs, higher-order structural information and consequently the memory of cell identity through cell division.
Cervical cancer cells express high-risk human papillomavirus (HPV) E6 and E7 proteins, and repression of HPV gene expression causes the cells to cease proliferation and undergo senescence. However, it is not known whether both HPV proteins are required to maintain the proliferative state of cervical cancer cells, or whether mutations that accumulate during carcinogenesis eliminate the need for one or the other of them. To address these questions, we used the bovine papillomavirus E2 protein to repress the expression of either the E6 protein or the E7 protein encoded by integrated HPV18 DNA in HeLa cervical carcinoma cells. Repression of the E7 protein activated the Rb pathway but not the p53 pathway and triggered senescence, whereas repression of the E6 protein activated the p53 pathway but not the Rb pathway and triggered both senescence and apoptosis. Telomerase activity, cyclin-dependent kinase activity, and expression of c-myc were markedly inhibited by repression of either E6 or E7. These results demonstrate that continuous expression of both the E6 and the E7 protein is required for optimal proliferation of cervical carcinoma cells and that the two viral proteins exert distinct effects on cell survival and proliferation. Therefore, strategies that inhibit the expression or activity of either viral protein are likely to inhibit the growth of HPV-associated cancers.
Infection with high-risk human papillomaviruses (HPVs) causes cervical cancer. E6 oncoprotein, an HPV gene product, inactivates the major gatekeeper p53. In contrast, its isoform, TAp73β, has become increasingly important, as it is resistant to E6. However, the intracellular signaling mechanisms that account for TAp73β tumor suppressor activity in cervix are poorly understood. Here, we identified that IER3 is a novel target gene of TAp73β. In particular, TAp73β exclusively transactivated IER3 in cervical cancer cells, whereas p53 and TAp63 failed to do. IER3 efficiently induced apoptosis, and its knockdown promoted survival of HeLa cells. In addition, TAp73β-induced cell death, but not p53-induced cell death, was inhibited upon IER3 silencing. Moreover, etoposide, a DNA-damaging chemotherapeutics, upregulated TAp73β and IER3 in a c-Abl tyrosine kinase-dependent manner, and the etoposide chemosensitivity of HeLa cells was largely determined by TAp73β-induced IER3. Of interest, cervical carcinomas from patients express no observable levels of two proteins. Thus, our findings suggest that IER3 is a putative tumor suppressor in the cervix, and the c-Ab1/p73β/IER3 axis is a novel and crucial signaling pathway that confers etoposide chemosensitivity. Therefore, TAp73β and IER3 induction would be a valuable checkpoint for successful therapeutic intervention of cervical carcinoma patients.
Deregulation of the Wnt/ β-catenin signal transduction pathway has been implicated in the pathogenesis of tumours in the mammary gland, colon and other tissues. Mutations in components of this pathway result in β-catenin stabilization and accumulation, and the aberrant modulation of β-catenin/TCF target genes. Such alterations in the cellular transcriptional profile are believed to underlie the pathogenesis of these cancers. We have sought to identify novel target genes of this pathway in mouse mammary epithelial cells.
Gene expression microarray analysis of mouse mammary epithelial cells inducibly expressing a constitutively active mutant of β-catenin was used to identify target genes of this pathway.
The differential expression in response to ΔNβ-catenin for five putative target genes, Autotaxin, Extracellular Matrix Protein 1 (Ecm1), CD14, Hypoxia-inducible gene 2 (Hig2) and Receptor Activity Modifying Protein 3 (RAMP3), was independently validated by northern blotting. Each of these genes encodes either a receptor or a secreted protein, modulation of which may underlie the interactions between Wnt/β-catenin tumour cells and between the tumour and its microenvironment. One of these genes, Hig2, previously shown to be induced by both hypoxia and glucose deprivation in human cervical carcinoma cells, was strongly repressed upon ΔNβ-catenin induction. The predicted N-terminus of Hig2 contains a putative signal peptide suggesting it might be secreted. Consistent with this, a Hig2-EGFP fusion protein was able to enter the secretory pathway and was detected in conditioned medium. Mutation of critical residues in the putative signal sequence abolished its secretion. The expression of human HIG2 was examined in a panel of human tumours and was found to be significantly downregulated in kidney tumours compared to normal adjacent tissue.
HIG2 represents a novel non-cell autonomous target of the Wnt pathway which is potentially involved in human cancer.
wnt; catenin; microarray; hypoxia; angiogenesis
Oral Cancer Overexpressed 1 (ORAOV1) is a candidate protooncogene locating on 11q13. Recent studies show that ORAOV1 acts as a primary driving force behind 11q13 gene amplification and plays a functional role in the tumorigenesis in a variety of human squamous cell carcinomas (SCCs). According to the results of molecular cytogenetic methods, 11q13 was characterized to be a high-level and recurrent amplification chromosomal site in cervical cancers. Up till now, the role of ORAOV1 in cervical cancer is unknown. The purpose of this study is to elucidate the function of ORAOV1 in cervical cancer cell growth by studying its roles in HeLa cells using small interfering RNA.
Functional analyses revealed that ORAOV1 was involved in the regulation of HeLa cell growth through its effect on cell cycle and apoptosis. Silence of ORAOV1 in HeLa cells downregulated the expression of Cyclin A, Cyclin B1 and Cdc2, and led to a distinct S cell cycle arrest. Moreover, knockdown of ORAOV1 expression activated both extrinsic and intrinsic apoptotic pathways and led to apoptosis in HeLa cells through its effect on the expression of several apoptosis related proteins such as P53, Bcl-2, Caspase-3, Caspase-8, Caspase-9 and cytochrome c. Interestingly, the expression of Cyclin D1, a pivotal gene for cervical cancer tumorigenesis, was also found to be reduced in ORAOV1 silenced HeLa cells.
Our findings indicate that ORAOV1 has an important role in regulating cell growth of cervical cancer HeLa cells through regulating the cell cycle and apoptosis. Thus, it may be a crucial protooncogene and a novel candidate therapeutic target for cervical cancer.