papiloma viruses "review on e6 and e7"

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SHERKO NASERI Advicer: Dr.Babak Behnam Cellular AND Molecular Biology Summer2011, Tehran University of medical science HPV ( E6,E7) : Cellular Mechanism of Interaction

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cellular and molecular behavior of E6 and E7 protein and oncogenesis

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Page 1: Papiloma Viruses "Review on E6 and E7"

SHERKO NASERIAdvicer: Dr.Babak Behnam

Cellular AND Molecular BiologySummer2011, Tehran University of medical science

HPV ( E6,E7) : Cellular Mechanism of Interaction

Page 2: Papiloma Viruses "Review on E6 and E7"

Cancer of the cervix

2nd most common cancer in women worldwide

Profiles like an STD (sexually transmitted disease) because of STD-dependent development

Importance

Page 3: Papiloma Viruses "Review on E6 and E7"

HPV is Necessary Cause of Cervical Cancer

Human papillomavirus DNA required for development of cervical cancer

HPV DNA detected in 90-100% of cervical cancer specimens compared to 5-20% in epidemiological control specimens

Bosch et al., 2002.

Page 4: Papiloma Viruses "Review on E6 and E7"

All characterized strains only infect epithelial cells, specifically skin anogenital mucosa oropharyngeal mucosa

HPV is Epitheliotropic

Human Papillomavirus model*

Page 5: Papiloma Viruses "Review on E6 and E7"

HPV Genome E1-E7 = “Early” genes

(nonstructural) L1, L2 = Capsid genes URR = upstream regulatory

region

E6 & E7 proteins play major role in immortality & malignant transformation of infected cells

E5 has role, but not required to maintain cancer phenotype

E2 Has a versus relate with E6 AND E7Munoz et al. 2006.

NORKIN,molecular virology,2008,ASM

Page 6: Papiloma Viruses "Review on E6 and E7"

HPV Classification & Carcinogenic Risk

Low Risk 60, 11, 42, 43, 44

Intermediate Risk

31, 33, 35, 51, 52, 58

High Risk 16, 18, 45, 56

Over 100 HPV strains identified

Risk assessment based on transformative potential of a strain’s E proteins

Low found in benign lesions only

Intermediate found in benign lesions & invasive cancers

High usually found in carcinomas; occasionally seen in benign lesions

Furumoto et al., 2002.

Page 7: Papiloma Viruses "Review on E6 and E7"

HPV’s E6 & E7 proteins interact with key cell cycle proteins including pRB & p53, effectively over-riding the G1/S-phase checkpoint

Mechanism1. E7 binds & phosphorylates pRB, activating E2F

transcription factor2. DNA replication proteins of host cell are then

expressed; unchecked S-phase occurs3. E6 marks p53 for proteolytic degradation so it

cannot activate apoptosis (note: absence of p53 is not necessary for E6 to cause immortalization)

Early Genes Hijack Cell Cycle Checkpoint

Page 8: Papiloma Viruses "Review on E6 and E7"
Page 9: Papiloma Viruses "Review on E6 and E7"

E6 & E7 in Cervical Cancer Progression

Furumoto et al., 2002.

Page 10: Papiloma Viruses "Review on E6 and E7"

Keratinocyte differentiation retarded

Checkpoint dependence goneChromosomal instability; accumulation of

oncogenic mutationsIncreased loss of cell cycle/growth control

Cancer

Consequences of the HPV Hijack

Page 11: Papiloma Viruses "Review on E6 and E7"

E6 CharacterizationA protein with 158 residues Four cysteine arrays—two

potential zinc fingers and molecular weight of about 18 kDa

N-terminal and C-terminal fragments encompassing residues 7-83 and 87-158

A short peptide at the C terminus of E6 interacts specifically with the PDZ domains of tumor suppressors

HPV-16 E6 protein from SWISS-Model Repository (P03126)

Page 12: Papiloma Viruses "Review on E6 and E7"

High-Risk HPV Oncoproteins: E6E6 Identified Function Investigator

(1) Cell immmortalization (2) Binding of E6-associated protein results in degradation of specific host cell proteins [p53] (3) Anti-apoptotic effect(4) Chromosomal destabilization (5) Enhancement of foreign DNA integration & mutagenicity (6) Activation of telomerase (7) Blockade of interferon functions(8) Degradation of Bak protein

(1) Band et al., 1990 (2) Werness et al., 1990 & Sheffner et al., 1993 (3) Werness et al., 1990 & Thomas, 1998 (4) White et al.,1994 (5) Kessis et al., 1996 & Havre et al., 1995 (6) Klingelhutz et al., 1996 (7) Ronco et al., 1998(8) Banks et al., 1998 & 1999

Hausen, 2000.

Page 13: Papiloma Viruses "Review on E6 and E7"

A ubiquitin thiolester cascade model for the HPV E6 dependent ubiquitination of p53

Ubiquitin is a 8 kDa protein of 76 amino-acid

Page 14: Papiloma Viruses "Review on E6 and E7"

E6BP and E6AP BindingE6 binding to cellular proteins can be

considered separately from p53 binding and degradation

for several reasons :1) E6 can bind to E6BP (ERC-55/E6BP) in the

absence of p532)BPV-1 E6 binds both E6BP and E6AP but

not p533) E6 binding to E6AP is a prerequisite for

p53 binding4) E6, E6BP and E6AP can form a ternary

complex

Page 15: Papiloma Viruses "Review on E6 and E7"
Page 16: Papiloma Viruses "Review on E6 and E7"

Stimulates expression of transcription factor HIF-1α

Both HPV E6 and E7 were able independently to enhance induction of HIF-1α upon DFO treatment. Enhancement of HIF-1α stability was not restricted to high risk HPV types, as HPV11, a low risk HPV type, mediated a similar effect(Mitsuhiro Nakamura et al,2010)

Prognostic Marker: Higher levels of HIF-1α expression in early-stage invasive cervical cancer correlated to shorter overall survival time

E6 Interact with HIF1-Alpha

Page 17: Papiloma Viruses "Review on E6 and E7"

In cells with normal functioning p53, HIF-1α is expressed in instances of hypoxia (as its name, hypoxia-inducible factor, implies)

HIF-1α binds & stabilizes p53 to induce apoptosis of hypoxic cells, however p53 is degraded by E6 in HPV-infected cells

Instead, HIF-1α stimulates neoangiogenesis for tumor cells, providing the vascularization necessary for cancer progression

Significance of HIF-1α Expression in Cervical Cancer

Page 18: Papiloma Viruses "Review on E6 and E7"

Hif-1α p53 BAX APOPTOSE

If P53 not present

Hif-1α

VEGF Neoangiogenesis

E6E7

Degradation

E6

Mdm2

Keyword:Mdm2(Mdm2 protein functions both as an E3 ubiquitin ligase that recognizes the N-terminal trans-activation domain (TAD) of the p53 tumor suppressor and an inhibitor of p53 transcriptional activation

Page 19: Papiloma Viruses "Review on E6 and E7"

Evidence of HIF-1α Overexpression in Cervical Cancer

No expression of HIF-1α in normal specimensAntibody treatment

less likely to disrupt normal cells

HIF-1α expression identified by nuclear staining/ immunohistochemistry

A

B

Invasive cervical cancer specimens exhibiting strong (A) & weak (B) HIF-1α expression

Birner et al., 2000

Page 20: Papiloma Viruses "Review on E6 and E7"
Page 21: Papiloma Viruses "Review on E6 and E7"

Candidate for Anti-angiogenesis Therapy?

Reduction of HIF-1α-induced angiogenesis may slow progression rate by cutting off oxygen & nutrient supply to tumor cells

HIF-1α mediates angiogenesis through activation of VEGF pathway Vascular endothelial

growth factor stimulates angiogenesis & release of similar factors

AntiHIF-1α or antiVEGF antibody treatment may control progression of cervical cancer

Page 22: Papiloma Viruses "Review on E6 and E7"

High-Risk HPV Oncoproteins: E7

E7 Identified Function Investigator(1) Cell immmortalization (2) Activation of cyclins D & E(3) Induction of apoptosis(4) Inhibition of cyclin-dependent kinase inhibitors(5) Enhancement of foreign DNA integration & mutagenicity (6) Degradation of Blk tyrosine kinase(7) Inactivation of retinoblastoma protein-related pocket proteins

(1) Munger & Phelps, 1993(2) Arroyo et al., 1993 & Zerfass et al., 1995 (3) Putthenveettil et al., 1996 (4) Jones et al., 1997 & Funk et al., 1997(5) Kessis et al., 1996 & Reznikoff et al., 1996 (6) Oda et al., 1999(7) Dyson et al., 1989, 1992.

Hausen, 2000.

Page 23: Papiloma Viruses "Review on E6 and E7"

E7 CharacterisationThe E7 proteins are small, acidic polypeptides

composed of approximately 100 amino acidsis its ability to bind and induce degradation of the

tumor-suppressor retinoblastoma protein (pRb) via the ubiquitin pathway

In the absent of pRb،E2f is separated from pRb and start to promote cell cycle

E7 contains two conserved regions (CR1,CR2)these two conserved regions significantly contribute

to the transforming activities of high-risk HPV E7 oncoproteins

Page 24: Papiloma Viruses "Review on E6 and E7"

Transcription factor Dp-1 is a protein that in humans is encoded by the TFDP1 gene

E2F factors bind to DNA as homodimers or heterodimers in association with dimerization partner DP1

Page 25: Papiloma Viruses "Review on E6 and E7"

E7 is PleiotropicInactivation of p21CIP-

1 & p27KIP-1 (cdk inhibitors) results in growth stimulation of infected cells

Inactivation of tumor suppressor transcription factor interferon 1 (IRF-1) through direct interaction HPV-1a E7 protein from

SWISS-Model Repository (P06465)

Page 26: Papiloma Viruses "Review on E6 and E7"

E7 Protein S tractureCR2 contains the LXCXE

domain (amino acids 22–26), which mediates the association with the pocket proteins, and two serines at positions 31 and 32, which are phosphorylated by casein kinase II. CR3 contains two CXXC motifs that are involved in zinc binding and in protein stabilization

Page 27: Papiloma Viruses "Review on E6 and E7"

IRF-1 Deactivation by E7

May explain the immune-resistance mechanism of HPV-infected cervical cancer cells

1. IRF-1 activated during exposure to viral infection, IFNs, TNFα, etc.

2. Histone deacetylase (HDAC) mediates accessibility to chromatin of IRF-1 inducible genes, such as IFN-β

3. IFN-β expression stimulates anti-proliferative effect on cell

Normal role of IRF-1 in tumor suppressor mechanism

Page 28: Papiloma Viruses "Review on E6 and E7"

E7 interacts with HDAC and IRF-1

E7 Zinc binding CR-3 is binding to histon deacetylase

CR3 contains two CXXC motifs that are involved in zinc binding

Blocks expression of IRF-1 inducible genes by inhibiting HDAC

Result: Cell proliferation evades immune response

Mechanism of IRF-1 Inactivation

Park et al., 2000.

Page 29: Papiloma Viruses "Review on E6 and E7"

Histone : Acetylation and Deacetylation

HDAC model – active domain with green

HADC caused to remove acetyl group (O=C-CH3) from an ε-N-acetyl lysine amino acid on a histone

DNA expression is regulated by acetylation and de-acetylation

If acetylation: histones displaced and DNA is accessible

If deacetilation acured DNA as is wrapped around histones

Page 30: Papiloma Viruses "Review on E6 and E7"

Notch1 expression would inhibit expression of HPV regulatory region (URR) & subsequent E6/E7 expression

Novel protective role against HPV-induced transformation

Notch1 Signaling Pathway in HPV-Cervical Cancer

Talora et al., 2002.

Page 31: Papiloma Viruses "Review on E6 and E7"

New Article in 2012

Human Papillomavirus type 16 (HPV-16) E7 and E6/E7 proteinsinhibited TNF-alpha-inducible NF-kB activity in human epithelial cells cultured

NF-kB influenced immortalization of cervical cells by HPV16

inhibition of NF-kB by HPV-16 E6/E7 contributes to immortalization of cells

Page 32: Papiloma Viruses "Review on E6 and E7"

New Article in 2011

We established two immortalized cell lines from human oral epithelium by transducing mutant cyclin dependent kinase 4, cyclin D1, and human telomerase reverse transcriptase with or without dominant-negative p53 into primary-cultured normal oral gingival epithelial cells using recombinant lentivirus vectors and named them MOE (mouth-ordinary-epithelium) 1a and MOE1b,respectively.

MOE1 cells kept the characteristics of normal epithelial cells without acquiring typical features of cancer cells and they could be useful not only for the study of oral neoplasm but also for other oral diseases

Page 33: Papiloma Viruses "Review on E6 and E7"

Bosch et al. 2002. The causal relationship between human papillomavirus and cervical cancer. J Clin Pathol 55:244-265.

Birner et al. 2000. Overexpression of Hypoxia-inducible Factor 1α Is a Marker for Unfavorable Prognosis in Early-stage Invasive Cervical Cancer. Cancer Research 60:4693-4696

Furumoto et al. 2002. Human papillomavirus (HPV) and cervical cancer. J Medical Investigation 49:124-122.

Hausen, H. 2000. Papillomaviruses Causing Cancer: Evasion from Host-Cell Control in Early Events in Carcinogenesis. J Natl Cancer Inst 92:690–8

Munoz et al. 2006. HPV in the etiology of human cancer. Vaccine 24S3:S3/1-S3/10

Park et al. 2000. Inactivation of Interferon Regulatory Factor-1 Tumor Suppressor Protein by HPV E7 Oncoprotein. J Bio Chem 275;10:6764-6769.

Talora et al. 2002. Specific down-modulation of Notch1 signaling in cervical cancer cells is required for sustained HPV-E6/E7 expression and late steps of malignant transformation. Genes & Dev 16:2252-2263

http://emc.medicines.org.uk/emc/assets/c/html/DisplayDoc.asp?DocumentID=19016

Toshiro Kibe et al.2011, Immortalization and characterization of normal oral epithelial cells withoutusing HPV and SV40 genes, Journal of the Japanese Stomatological Society,14 mrch2011

Erik R. Vandermark et al.2011, Human papillomavirus type 16 E6 and E 7 proteins alter NF-kB in cultured cervical epithelial cells and inhibition of NF-kB promotes cell growth and immortalization,28jan2012

References

Page 34: Papiloma Viruses "Review on E6 and E7"