SHERKO NASERI Advicer: Dr.

Babak Behnam Cellular AND Molecular Biology Summer2011, Tehran University of medical science

 Cancer of the cervix

2nd most common cancer in women worldwide Profiles like an STD (sexually transmitted disease)

because of STD-dependent development

 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.

 All characterized strains only infect epithelial cells,

specifically

skin anogenital mucosa oropharyngeal mucosa

Human Papillomavirus model*

 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 E7
Munoz et al. 2006. NORKIN,molecular virology,2008,ASM

 Over 100 HPV strains

identified Risk assessment based on transformative potential of a strains E proteins

Low Risk 60, 11, 42, 43, 44

Low found in benign

Intermediate 31, 33, 35, 51, 52, Risk 58

lesions only Intermediate found in benign lesions & invasive cancers High usually found in carcinomas; occasionally seen in benign lesions

High Risk 16, 18, 45, 56

Furumoto et al., 2002.

HPVs E6 & E7 proteins interact with key cell cycle proteins including pRB & p53, effectively over-riding the G1/S-phase checkpoint Mechanism 1. E7 binds & phosphorylates pRB, activating E2F transcription factor 2. DNA replication proteins of host cell are then expressed; unchecked S-phase occurs 3. E6 marks p53 for proteolytic degradation so it cannot activate apoptosis (note: absence of p53 is not necessary for E6 to cause immortalization)

Furumoto et al., 2002.

 Keratinocyte differentiation retarded Checkpoint dependence gone

Chromosomal instability; accumulation of oncogenic

mutations Increased loss of cell cycle/growth control

Cancer

 A protein with 158 residues

Four cysteine arraystwo 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)

E6 Identified Function
(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

Investigator
(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.

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

Ubiquitin is a 8 kDa protein of 76 amino-acid

 E6 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 p53 2)BPV-1 E6 binds both E6BP and E6AP but not p53 3) E6 binding to E6AP is a prerequisite for p53 binding 4) E6, E6BP and E6AP can form a ternary complex

 Stimulates expression of transcription factor HIF-

1 Both HPV E6 and E7 were able independently to enhance induction of HIF-1 upon DFO treatment. Enhancement of HIF-1 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-1 expression in early-stage invasive cervical cancer correlated to shorter overall survival time

 In cells with normal functioning p53, HIF-1 is

expressed in instances of hypoxia (as its name, hypoxia-inducible factor, implies) HIF-1 binds & stabilizes p53 to induce apoptosis of hypoxic cells, however p53 is degraded by E6 in HPV-infected cells Instead, HIF-1 stimulates neoangiogenesis for tumor cells, providing the vascularization necessary for cancer progression

Mdm2 Degradation E6

Hif-1

p53

BAX

APOPTOSE

If P53 not present

Hif-1

VEGF

Neoangiogenesis

E6

E7

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

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

No expression of HIF-1

in normal specimens
Antibody treatment less

likely to disrupt normal cells

HIF-1 expression

identified by nuclear staining/ immunohistochemistry

Birner et al., 2000

 HIF-1 mediates

Reduction of HIF-1-

induced angiogenesis may slow progression rate by cutting off oxygen & nutrient supply to tumor cells

angiogenesis through activation of VEGF pathway

Vascular endothelial growth

factor stimulates angiogenesis & release of similar factors AntiHIF-1 or antiVEGF antibody treatment may control progression of cervical cancer

E7 Identified Function
(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

Investigator
(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.

 The E7 proteins are small, acidic polypeptides composed

of approximately 100 amino acids is its ability to bind and induce degradation of the tumorsuppressor retinoblastoma protein (pRb) via the ubiquitin pathway In the absent of pRbE2f 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

 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

 Inactivation 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)

 CR2 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

 May explain the

immuneresistance mechanism of HPV-infected cervical cancer cells Normal role of

IRF-1 in tumor suppressor mechanism

1. IRF-1 activated during exposure to viral infection, IFNs, TNF, etc. 2. Histone deacetylase (HDAC) mediates accessibility to chromatin of IRF-1 inducible genes, such as IFN- 3. IFN- expression stimulates antiproliferative effect on cell

 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

Park et al., 2000.

 HADC caused to remove acetyl

HDAC model active domain with green

group (O=C-CH3) from an -Nacetyl 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

 Notch1 expression

would inhibit expression of HPV regulatory region (URR) & subsequent E6/E7 expression Novel protective role against HPVinduced transformation

Talora et al., 2002.

Human Papillomavirus type 16 (HPV-16) E7 and E6/E7 proteins inhibited 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

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

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 1 Is a Marker for Unfavorable Prognosis in Early-stage Invasive Cervical Cancer. Cancer Research 60:46934696 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 92:6908 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