scholarly journals CRISPR/Cas9-based inactivation of human papillomavirus oncogenes E6 and E7 induces senescence in cervical cancer cells

2020 ◽  
Author(s):  
Raviteja Inturi ◽  
Per Jemth
Keyword(s):  
Cancer Cells ◽  
Cellular Senescence ◽  
Hela Cells ◽  
Human Papillomaviruses ◽  
Alternative Outcome ◽  
Cervical Cancer Cells ◽  
Nucleus Surface ◽  
Hpv18 E6 ◽  
E6 And E7 ◽  
E7 Proteins

ABSTRACTHuman papillomaviruses (HPVs) such as HPV16 and HPV18 can cause cancers of the cervix, vagina, vulva, penis, anus and oropharynx. Continuous expression of the HPV viral oncoproteins E6 and E7 are essential for transformation and maintenance of cancer cells. Therefore, therapeutic targeting of E6 and E7 genes can potentially be used to treat HPV-related cancers. Previous CRISPR/Cas9 studies on inactivation of E6 and E7 genes confirmed cell cycle arrest and apoptosis. Here we report that CRISPR/Cas9-based knockout of E6 and E7 can also trigger cellular senescence in HPV18 immortalized HeLa cells. Specifically, HeLa cells in which E6 and E7 were inactivated exhibited characteristic senescence markers like enlarged cell and nucleus surface area, increased β-galactosidase expression, and loss of lamin B1 with detection of cytoplasmic chromatin fragments. Furthermore, the knockout of HPV18 E6 and E7 proteins resulted in upregulation of p53/p21 and pRb/p21 levels in senescent cells. These senescent cells were devoid of characteristic apoptotic markers and re-introduction of codon-modified HPV18 E6 decreased p53 levels. Taken together, our study demonstrates that cellular senescence is as an alternative outcome of HPV oncogene inactivation by the CRISPR/Cas9 methodology.

scholarly journals Endogenous Human Papillomavirus E6 and E7 Proteins Differentially Regulate Proliferation, Senescence, and Apoptosis in HeLa Cervical Carcinoma Cells

2003 ◽  
Vol 77 (2) ◽  
pp. 1551-1563 ◽  
Author(s):  
Rosa Anna DeFilippis ◽  
Edward C. Goodwin ◽  
Lingling Wu ◽  
Daniel DiMaio
Keyword(s):  
Cervical Cancer ◽  
Human Papillomavirus ◽  
Cancer Cells ◽  
Cervical Carcinoma ◽  
Carcinoma Cells ◽  
Rb Pathway ◽  
Cervical Cancer Cells ◽  
E6 And E7 ◽  
E7 Proteins ◽  
E6 Protein

ABSTRACT 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.

scholarly journals Human papillomavirus type 18 oncoproteins exert their oncogenicity in esophageal and tongue squamous cell carcinoma cell lines distinctly

2019 ◽  
Author(s):  
Siaw Shi Boon ◽  
Zigui Chen ◽  
Karen Lee Yin Ching ◽  
Liuyang Cai ◽  
Jintao Li ◽  
...  
Keyword(s):  
Cervical Cancer ◽  
Cancer Cells ◽  
Cell Lines ◽  
Cancer Cell ◽  
Tongue Cancer ◽  
Cancer Cell Lines ◽  
Cervical Cancer Cells ◽  
Esophageal Cancer Cell ◽  
Hpv18 E6 ◽  
E6 And E7

Abstract Background: Increasing evidence indicates an etiological role of human papillomavirus (HPV) in head and neck cancers, particularly oropharyngeal squamous cell carcinoma (OPSCC). However, the association between HPV and other cancers, including esophageal and tongue remains unclear. Methods: We compared the molecular characteristics of HPV18 E6 and E7 in esophageal (EC109 and EC9706) and tongue (Tca83) cancer cell lines with reference to cervical cancer (HeLa). We studied the expression of HPV transcripts using Next Generation RNA sequencing. We used small interference RNA (siRNA) against HPV18 oncoproteins to verify their oncogenic roles in molecular signaling, targeting metalloproteases and apoptotic pathways in these cancers. Results: We observed that the HPV transcription profiles of esophageal and tongue cancer cells mimicked that of cervical cancer cells, with notable disruption of E2, and expression of E6, spliced E6 (E6 * ), E7, E1 and L1 transcripts. As with cervical cancer cells, p53 and its downstream transactivation target, p21, were found to be the major targets of E6 in esophageal and tongue cancer cell lines. Intriguingly, E7 preferentially targeted p130 in the two esophageal cancer cell lines, instead of pRb as in cervical cancer. Tca83 exhibited an E7 to E6 transcript ratio comparable to HeLa (cervix), targeted the ERK1/2 and MMP2 pathways, and was dependent on E6 and E7 to survive and proliferate. In contrast, both the esophageal cancer cell lines were distinct from HeLa in these aspects. Conclusion: This is the first study to delineate the transcripts expression and role of HPV18 E6 and E7 in esophageal and tongue cancer cell lines. This findings suggest that HPV18 plays an oncogenic role in inducing these cancers, albeit via distinct pathways than those observed in cervical cancer.

scholarly journals E6-mediated activation of JNK drives EGFR signalling to promote proliferation and viral oncoprotein expression in cervical cancer

2020 ◽  
Author(s):  
Ethan L. Morgan ◽  
James A. Scarth ◽  
Molly R. Patterson ◽  
Christopher W. Wasson ◽  
Georgia C. Hemingway ◽  
...  
Keyword(s):  
Cervical Cancer ◽  
Cell Proliferation ◽  
Cancer Cells ◽  
Human Papillomaviruses ◽  
Signalling Pathway ◽  
Viral Oncoprotein ◽  
Hpv E6 ◽  
Cervical Cancer Cells ◽  
E6 And E7 ◽  
Novel Therapeutic

AbstractHuman papillomaviruses (HPV) are a major cause of malignancy worldwide, contributing to ~5% of all human cancers including almost all cases of cervical cancer and a growing number of ano-genital and oral cancers. HPV-induced malignancy is primarily driven by the viral oncogenes, E6 and E7, which manipulate host cellular pathways to increase cell proliferation and enhance cell survival, ultimately predisposing infected cells to malignant transformation. Consequently, a more detailed understanding of viral-host interactions in HPV-associated disease offers the potential to identify novel therapeutic targets. Here, we identify that the c-Jun N-terminal kinase (JNK) signalling pathway is activated in cervical disease and in cervical cancer. The HPV E6 oncogene induces JNK1/2 phosphorylation in a manner that requires the E6 PDZ binding motif. We show that blockade of JNK1/2 signalling using small molecule inhibitors, or knockdown of the canonical JNK substrate c-Jun, reduces cell proliferation and induces apoptosis in cervical cancer cells. We further demonstrate that this phenotype is at least partially driven by JNK-dependent activation of EGFR signalling via increased expression of EGFR and the EGFR ligands EGF and HB-EGF. JNK/c-Jun signalling promoted the invasive potential of cervical cancer cells and was required for the expression of the epithelial to mesenchymal transition (EMT)-associated transcription factor Slug and the mesenchymal marker Vimentin. Furthermore, JNK/c-Jun signalling is required for the constitutive expression of HPV E6 and E7, which are essential for cervical cancer cell growth and survival. Together, these data demonstrate a positive feedback loop between the EGFR signalling pathway and HPV E6/E7 expression, identifying a regulatory mechanism in which HPV drives EGFR signalling to promote proliferation, survival and EMT. Thus, our study has identified a novel therapeutic target that may be beneficial for the treatment of cervical cancer.

scholarly journals Repression of the Integrated Papillomavirus E6/E7 Promoter Is Required for Growth Suppression of Cervical Cancer Cells

2000 ◽  
Vol 74 (6) ◽  
pp. 2679-2686 ◽  
Author(s):  
Delicia A. Francis ◽  
Susanne I. Schmid ◽  
Peter M. Howley
Keyword(s):  
Cancer Cells ◽  
Hela Cells ◽  
Cellular Proliferation ◽  
Growth Arrest ◽  
Bovine Papillomavirus ◽  
Transactivation Domain ◽  
Hpv16 E6 ◽  
Cervical Cancer Cells ◽  
E6 And E7 ◽  
High Risk Hpv

ABSTRACT The human papillomavirus (HPV) E2 protein is an important regulator of viral E6 and E7 gene expression. E2 can repress the viral promoter for E6 and E7 expression as well as block progression of the cell cycle in cancer cells harboring the DNA of “high-risk” HPV types. Although the phenomenon of E2-mediated growth arrest of HeLa cells and other HPV-positive cancer cells has been well documented, the specific mechanism by which E2 affects cellular proliferation has not yet been elucidated. Here, we show that bovine papillomavirus (BPV) E2-induced growth arrest of HeLa cells requires the repression of the E6 and E7 promoter. This repression is specific for E2TA and not E2TR, a BPV E2 variant that lacks the N-terminal transactivation domain. We demonstrate that expression of HPV16 E6 and E7 from a heterologous promoter that is not regulated by E2 rescues HeLa cells from E2-mediated growth arrest. Our data indicate that the pathway of E2-mediated growth arrest of HeLa cells requires repression of E6 and E7 expression through an activity specified by the transactivation domain of E2TA.

scholarly journals Disruption of repressive p130–DREAM complexes by human papillomavirus 16 E6/E7 oncoproteins is required for cell-cycle progression in cervical cancer cells

2011 ◽  
Vol 92 (11) ◽  
pp. 2620-2627 ◽  
Author(s):  
Nurshamimi Nor Rashid ◽  
Rohana Yusof ◽  
Roger J. Watson
Keyword(s):  
Cell Cycle ◽  
Cervical Cancer ◽  
Cancer Cells ◽  
Cell Cycle Progression ◽  
Human Papillomaviruses ◽  
G1 Arrest ◽  
Cycle Progression ◽  
Hpv16 E6 ◽  
Cervical Cancer Cells ◽  
E6 And E7

Human papillomaviruses (HPVs) with tropism for mucosal epithelia are the major aetiological factors in cervical cancer. Most cancers are associated with so-called high-risk HPV types, in particular HPV16, and constitutive expression of the HPV16 E6 and E7 oncoproteins is critical for malignant transformation in infected keratinocytes. E6 and E7 bind to and inactivate the cellular tumour suppressors p53 and Rb, respectively, thus delaying differentiation and inducing proliferation in suprabasal keratinocytes to enable HPV replication. One member of the Rb family, p130, appears to be a particularly important target for E7 in promoting S-phase entry. Recent evidence indicates that p130 regulates cell-cycle progression as part of a large protein complex termed DREAM. The composition of DREAM is cell cycle-regulated, associating with E2F4 and p130 in G0/G1 and with the B-myb transcription factor in S/G2. In this study, we addressed whether p130–DREAM is disrupted in HPV16-transformed cervical cancer cells and whether this is a critical function for E6/E7. We found that p130–DREAM was greatly diminished in HPV16-transformed cervical carcinoma cells (CaSki and SiHa) compared with control cell lines; however, when E6/E7 expression was targeted by specific small hairpin RNAs, p130–DREAM was reformed and the cell cycle was arrested. We further demonstrated that the profound G1 arrest in E7-depleted CaSki cells was dependent on p130–DREAM reformation by also targeting the expression of the DREAM component Lin-54 and p130. The results show that continued HPV16 E6/E7 expression is necessary in cervical cancer cells to prevent cell-cycle arrest by a repressive p130–DREAM complex.

scholarly journals Human Papillomavirus E6/E7 and Long Noncoding RNA TMPOP2 Mutually Upregulated Gene Expression in Cervical Cancer Cells

2019 ◽  
Vol 93 (8) ◽  
Author(s):  
Hongpeng He ◽  
Xiang Liu ◽  
Yue Liu ◽  
Mengmeng Zhang ◽  
Yongwei Lai ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Cervical Cancer ◽  
Human Papillomavirus ◽  
Cancer Cells ◽  
Long Noncoding Rna ◽  
Noncoding Rna ◽  
Human Papillomaviruses ◽  
Cervical Cancer Cells ◽  
E6 And E7

ABSTRACT TMPOP2 was previously suggested to be an oncogenic long noncoding RNA which is excessively expressed in cervical cancer cells and inhibits E-cadherin gene expression by recruiting transcription repressor EZH2 to the gene promoter. So far, the function and regulation of TMPOP2 in cervical cancer remain largely unknown. Herein, we found that TMPOP2 expression was correlated with human papillomavirus 16/18 (HPV16/18) E6 and E7 in cervical cancer cell lines CaSki and HeLa. Tumor suppressor p53, which is targeted for degradation by HPV16/18, was demonstrated to associate with two p53 response elements in the TMPOP2 promoter to repress the transcription of the TMPOP2 gene. Reciprocally, ectopic expression of TMPOP2 was demonstrated to sequester tumor repressor microRNAs (miRNAs) miR-375 and miR-139 which target HPV16/18 E6/E7 mRNA and resulted in an upregulation of HPV16/18 E6/E7 genes. Thereby, HPV16/18 E6/E7 and the long noncoding RNA (lncRNA) TMPOP2 form a positive feedback loop to mutually derepress gene expression in cervical cancer cells. Moreover, results of RNA sequencing and cell cycle analysis showed that knockdown of TMPOP2 impaired the expression of cell cycle genes, induced cell cycle arrest, and inhibited HeLa cell proliferation. Together, our results indicate that TMPOP2 and HPV16/18 E6/E7 mutually strengthen their expression in cervical cancer cells to enhance tumorigenic activities. IMPORTANCE Human papillomaviruses 16 and 18 (HPV16/18) are the main causative agents of cervical cancer. Viral proteins HPV16/18 E6 and E7 are constitutively expressed in cancer cells to maintain oncogenic phenotypes. Accumulating evidences suggest that HPVs are correlated with the deregulation of long noncoding RNAs (lncRNAs) in cervical cancer, although the mechanism was unexplored in most cases. TMPOP2 is a newly identified lncRNA excessively expressed in cervical cancer. However, the mechanism for the upregulation of TMPOP2 in cervical cancer cells remains largely unknown and its relationship with HPVs is still elusive. The significance of our research is in revealing the mutual upregulation of HPV16/18 E6/E7 and TMPOP2 with the molecular mechanisms explored. This study will expand our understandings of the oncogenic activities of human papillomaviruses and lncRNAs.

scholarly journals Human papillomavirus type 18 oncoproteins exert their oncogenicity in esophageal and tongue squamous cell carcinoma cell lines distinctly

2019 ◽  
Author(s):  
Siaw Shi Boon ◽  
Zigui Chen ◽  
Jintao Li ◽  
Karen Lee Yin Ching ◽  
Liuyang Cai ◽  
...  
Keyword(s):  
Cervical Cancer ◽  
Cancer Cells ◽  
Cell Lines ◽  
Cancer Cell ◽  
Tongue Cancer ◽  
Cancer Cell Lines ◽  
Cervical Cancer Cells ◽  
Esophageal Cancer Cell ◽  
Hpv18 E6 ◽  
E6 And E7

Abstract Increasing evidence indicates an etiological role of human papillomavirus (HPV) in head and neck cancers, particularly oropharyngeal squamous cell carcinoma (OPSCC). However, the association between HPV and other cancers, including esophageal and tongue remains unclear. This study delineated the molecular characteristics of HPV18 E6 and E7 in esophageal (EC109 and EC9706) and tongue (Tca83) cancer cell lines with reference to cervical cancer (HeLa). Overall, the HPV transcription profiles of esophageal and tongue cancer cells mimicked that of cervical cancer cells, with notable disruption of E2, and expression of E6, spliced E6 (E6*), E7, E1 and L1 transcripts. As with cervical cancer cells, p53 and its downstream transactivation target, p21, were found to be the major targets of E6 in esophageal and tongue cancer cell lines. Intriguingly, E7 preferentially targeted p130 in the two esophageal cancer cell lines, instead of pRb as in cervical cancer. Tca83 exhibited an E7 to E6 transcript ratio comparable to HeLa (cervix), targeted the ERK1/2 and MMP2 pathways, and was dependent on E6 and E7 to survive and proliferate. In contrast, both the esophageal cancer cell lines were distinct from HeLa in these aspects. In conclusion, this is the first study that delineates transcript expression and protein interaction of HPV18 E6 and E7 in esophageal and tongue cancer cell lines, suggesting that HPV plays a role in inducing these cancers, albeit via distinct pathways than those observed in cervical cancer.

Reducing invasion potential of cervical cancer cells via targeted knockdown of c-Jun.

2013 ◽  
Vol 31 (15_suppl) ◽  
pp. e22005-e22005
Author(s):  
Grace Pei Chien Yee ◽  
Paul L. De Souza ◽  
Levon M. Khachigian
Keyword(s):  
Cervical Cancer ◽  
Cell Proliferation ◽  
Cancer Cells ◽  
Hela Cells ◽  
Human Papillomaviruses ◽  
Mrna Levels ◽  
Down Regulation ◽  
Recurrent Cervical Cancer ◽  
Invasion Potential ◽  
Cervical Cancer Cells

e22005 Background: Despite recent advent of vaccines for human papillomaviruses (HPV) in cervical cancer and increasing efforts to improve therapy, deaths still average 275,000 annually worldwide, with most women succumbing to recurrent or metastatic disease. The c-Jun oncogene is a subunit of the activating protein-1 (AP-1) transcription factor and is strongly expressed in cervical cancer, regulating the expression of HPV16 and 18 genes. AP-1 plays a major role in cell growth, migration and apoptosis in many cell types. This study examined the role of c-Jun in modulating HeLa proliferation, migration, apoptosis and invasion as well as susceptibility to cisplatin. Methods: Transient knockdown and over-expression of c-Jun was performed in HeLa cervical cancer cells using Dharmacon c-Jun siRNA and Origene Jun expression vector. c-Jun and downstream gene/protein expression was confirmed by western blot and real-time PCR and cells subject to proliferation, in vitro wound and matrigel dual-chamber transwell assays. Flow cytometry was used for analysis of cell cycle and apoptosis. Results: c-Jun protein and mRNA levels were reduced by c-Jun siRNA. c-Jun silencing inhibited cervical cancer cell proliferation. Significantly, c-Jun suppression dramatically reduced HeLa migration and invasion and targeted down-regulation of cyclooxygenase-2 (Cox2), interleukin-6 (IL-6), metalloproteinases (MMP)-1, -9 and -13 as well as HPV18 E6 and E7, genes highly expressed in cervical cancer and associated with metastatic growth. Direct siRNA knockdown of Cox2 in HeLa also reduced MMP1 and MMP9 expression suggesting an intermediary link. In HeLa cells over-expressing c-Jun, cell proliferation was not significantly increased but cell invasiveness was markedly enhanced in parallel with enhanced MMP-1 expression. Modulation of c-Jun expression did not interfere with susceptibility of HeLa cells to apoptosis in the presence of cisplatin. Conclusions: Reduced invasion potential of HeLa cells after c-Jun knockdown suggests a potential target in treatment of metastatic and recurrent cervical cancer. Data suggest a mechanism involving down-regulation of Cox2 and MMP-1 and -9 expression.

scholarly journals Nucleolin as Activator of Human Papillomavirus Type 18 Oncogene Transcription in Cervical Cancer

2002 ◽  
Vol 196 (8) ◽  
pp. 1067-1078 ◽  
Author(s):  
Edgar Grinstein ◽  
Peter Wernet ◽  
Peter J.F. Snijders ◽  
Frank Rösl ◽  
Inge Weinert ◽  
...  
Keyword(s):  
Cervical Cancer ◽  
High Risk ◽  
Cancer Cells ◽  
Binding Activity ◽  
Human Papillomaviruses ◽  
Cervix Uteri ◽  
Cell Protein ◽  
Critical Event ◽  
Cervical Cancer Cells ◽  
E6 And E7

High risk human papillomaviruses (HPVs) are central to the development of cervical cancer and the deregulated expression of high risk HPV oncogenes is a critical event in this process. Here, we find that the cell protein nucleolin binds in a sequence-specific manner to the HPV18 enhancer. The DNA binding activity of nucleolin is primarily S phase specific, much like the transcription of the E6 and E7 oncoproteins of HPV18 in cervical cancer cells. Antisense inactivation of nucleolin blocks E6 and E7 oncogene transcription and selectively decreases HPV18+ cervical cancer cell growth. Furthermore, nucleolin controls the chromatin structure of the HPV18 enhancer. In contrast, HPV16 oncogene transcription and proliferation rates of HPV16+ SiHa cervical cancer cells are independent of nucleolin activity. Moreover, nucleolin expression is altered in HPV18+ precancerous and cancerous tissue from the cervix uteri. Whereas nucleolin was homogeneously distributed in the nuclei of normal epithelial cells, it showed a speckled nuclear phenotype in HPV18+ carcinomas. Thus, the host cell protein nucleolin is directly linked to HPV18-induced cervical carcinogenesis.

scholarly journals Stabilization of p53 by microRNAs in HPV-positive cervical cancer cells

2020 ◽  
Author(s):  
Gustavo Martínez-Noël ◽  
Patricia Szajner ◽  
Jennifer A. Smith ◽  
Kathleen Boyland ◽  
Rebecca E. Kramer ◽  
...  
Keyword(s):  
Cervical Cancer ◽  
Cancer Cells ◽  
High Throughput ◽  
Ubiquitin Ligase ◽  
Hela Cells ◽  
P53 Protein ◽  
Human Papillomaviruses ◽  
Live Cell ◽  
Protein Levels ◽  
Cervical Cancer Cells

AbstractEtiologically 5% of cancers worldwide are caused by the high-risk human papillomaviruses (hrHPVs). These viruses encode two oncoproteins (E6 and E7) whose expression is required for cancer initiation and maintenance. Among the cellular targets of these viral oncoproteins are the p53 and the retinoblastoma tumor suppressor proteins. Inhibition of E6-mediated ubiquitylation of p53 through the E6AP ubiquitin ligase results in the stabilization of p53, leading to cellular apoptosis. We utilized a live cell high-throughput screen to assess the ability of 885 microRNAs (miRNAs) to stabilize p53 in human papillomavirus (HPV)-positive cervical cancer cells expressing a p53-fluorescent protein as an in vivo reporter of p53 stability. The 32 miRNAs whose expression resulted in the greatest p53 stabilization were further assessed in validation experiments using a second cell-based p53 stability reporter system as well as in HeLa cells to examine their effects on endogenous p53 protein levels. The positive miRNAs identified included 375-3p that has previously been reported as stabilizing p53 in HeLa cells, providing validation of the screen. Additional miRNAs that stabilized p53 led to decreases in E6AP protein levels, while others, including members of the 302/519 family of miRNAs, targeted HPV oncoprotein expression. We further examined a subset of these miRNAs for their abilities to induce apoptosis and determined whether the apoptosis was p53-mediated. The miRNAs described here have potential as therapeutics for treating HPV-positive cancers.Author summaryHuman papillomaviruses cause approximately 5% of cancers worldwide and encode genes that contribute to both the initiation and maintenance of these cancers. The viral gene E6 is expressed in all HPV-positive cancers and functions by targeting the degradation of the p53 protein through its engagement of the cellular ubiquitin ligase E6AP. Inhibiting the degradation of p53 results in apoptosis in HPV-positive cancer cells. We have developed a high-throughput live cell assay to identify molecules that stabilize p53 in HPV-positive cells and we present the results of a screen we have carried out examining miRNAs for their abilities to stabilize p53 and induce apoptosis in HPV-positive cervical cancer cells. These miRNAs have the potential to be used in the treatment of HPV-positive cancers.

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