molecular pathogenesis of cervical cancer

8/16/2019 Molecular Pathogenesis of Cervical Cancer

1/13

Full Terms & Conditions of access and use can be found athttp://www.tandfonline.com/action/journalInformation?journalCode=kcbt20

Download by: [115.178.196.97] Date: 31 May 2016, At: 18:17

Cancer Biology & Therapy

ISSN: 1538-4047 (Print) 1555-8576 (Online) Journal homepage: http://www.tandfonline.com/loi/kcbt20

Molecular pathogenesis of cervical cancer

Okechukwu A. Ibeanu

To cite this article: Okechukwu A. Ibeanu (2011) Molecular pathogenesis of cervical cancer ,Cancer Biology & Therapy, 11:3, 295-306, DOI: 10.4161/cbt.11.3.14686

To link to this article: http://dx.doi.org/10.4161/cbt.11.3.14686

Published online: 01 Feb 2011.

Submit your article to this journal

Article views: 1951

View related articles

Citing articles: 20 View citing articles

http://www.tandfonline.com/doi/citedby/10.4161/cbt.11.3.14686#tabModulehttp://www.tandfonline.com/doi/citedby/10.4161/cbt.11.3.14686#tabModulehttp://www.tandfonline.com/doi/mlt/10.4161/cbt.11.3.14686http://www.tandfonline.com/doi/mlt/10.4161/cbt.11.3.14686http://www.tandfonline.com/action/authorSubmission?journalCode=kcbt20&page=instructionshttp://www.tandfonline.com/action/authorSubmission?journalCode=kcbt20&page=instructionshttp://dx.doi.org/10.4161/cbt.11.3.14686http://www.tandfonline.com/action/showCitFormats?doi=10.4161/cbt.11.3.14686http://www.tandfonline.com/loi/kcbt20http://www.tandfonline.com/action/journalInformation?journalCode=kcbt20


2/13

www.landesbioscience.com Cancer Biology & Therapy 295

Cancer Biology & Therapy 11:3, 295-306; February 1, 2011; © 2011 Landes Bioscience

REVIEW REVIEW

Introduction

Scientic studies over the past few decades have provided

denitive evidence that cervical cancer is a sexually transmit-ted disease that results from infection with certain high-risk,oncogenic types of the human papillomavirus. HPV DNAis found in 99% of invasive cervical cancers worldwide.1,2 Furthermore, vaccination aga inst HPV prevents the acquisitionof the precursor lesion of cervical cancer, high-grade squamousintraepithelial lesions (HSILs), also termed high grade cervicalintraepithelial neoplasia (CIN2/3). While successful imple-mentation of cervical cancer screening protocols and treat-ment of HSIL have led to a decline in mortality in developedareas of the world, cervical cancer continues to be a leadingcause of cancer related deaths in developing countries due to acombination of the high prevalence of HPV infection and the

lack of widespread availability of cervical Papanicolau smear(Pap smear) testing of susceptible women.3 Molecular stud-ies of HPV carcinogenesis have revealed fundamental mecha-nisms by which HPV replicates, transforms cells and evadesthe immune system. Extensive epidemiologic data and clinicaltrials have led to renements in screening, including new clini-cal tests for oncogenic HPV infection and treatment protocols

Correspondence to: Okechukwu A. Ibeanu; Email: [email protected]: 12/06/10; Accepted: 12/30/10DOI: 10.4161/cbt.11.3.14686

Introduction: Cervical cancer is a sexually transmitted diseasethat results from infection with oncogenic types of humanpapillomavirus (HPV). Oncogenic HPV DNA is found in over95% of invasive cervical cancers worldwide. Cervical canceris a leading cause of cancer deaths in developing countriesbecause of high HPV infection rates and lack of comprehensivecervical Pap smear testing of susceptible women. Vaccinationagainst HPV prevents the acquisition of cervical dysplasticlesions among eligible women who have not already acquiredthe vaccine-specic HPV types.

Methods: Literature review of the epidemiology, infection

cycle, viral gene function and current vaccines related to theoncogenic types of the human papillomavirus (HPV).

Conclusion: Enhanced understanding of HPV and popula-tion-based measures offer the best hope of limiting worldwidemortality due to cervical cancer. The development of thera-peutic cervical cancer vaccines and/or virus-targeted drugtherapies would be a giant step forward.

Molecular pathogenesis of cervical cancerOkechukwu A. Ibeanu

Division of Gynecologic Oncology; Post-Doctoral Fellow; Johns Hopkins University; Baltimore, MD USA

Key words: HPV, cervix, carcinogenesis, dysplasia, early, gene, vaccine

based on a better understanding of the natural course of HPVinfection and accompanying cervical premalignant disease.

This review aims to discuss the pathogenesis and immuno-logic changes of HPV-related cervical cancer as well as the clini-cal course of HPV infection in relation to current screening andtreatment guidelines. The role of the HPV early proteins willbe discussed as well as current clinical management of cervicaldysplasia.

Epidemiology of Human Papilloma Virus

Cervical cancer is the second most prevalent cancer seen in women worldwide, with about 500,000 cases and over 270,000deaths estimated annually.3,4 Sub-Saharan Africa, South Americaand Southeast Asia have the highest prevalence of HPV with 25,15 and 8% of women infected, respectively.3 In the United States,cervical cancer is the 12th most common cancer in women with11,000 cases and 3,500 deaths reported in 2008.5 The incidenceof cervical cancer in the United States has dropped by about 90%over the past half century due to effective implementation ofcervical cancer screening and treatment protocols. Nonetheless,considering that 80% of sexually active US adults will acquireHPV during their lifetimes,4 HPV-related diseases will continue

to impose a considerable burden on healthcare resources for theforeseeable future. Fortunately, the recent licensure of two pre-ventive HPV vaccines provides an important opportunity toeventually eliminate cervical cancer upon comprehensive vacci-nation prior to sexual debut.

The central etiologic factor for the development of cervicalcancer is persistent infection with high-risk oncogenic HPVtypes.6 Other recognized risk factors for cervical cancer arerelated to the sexual acquisition of HPV as well as immune dys-function, exposure to mutagens and hormonal factors. Studiesin twins suggest that genetic background typically plays a smallrole in the development of cervical cancer. The most notablerisks are early age of sexual activity, multiple sexual partners,

exposure to other sexually transmitted diseases, cigarette smok-ing, oral contraceptive use, human immunodeciency virusinfection and immunosuppressive drug therapy.3 Poor accessto cancer screening services as well as non-compliance withscreening visits are social risk factors related to lower socio-economic status and lower educational levels that are observedmore frequently among African American, American Indianand Hispanic women compared to Caucasians in the UnitedStates.3 Unfortunately, these same factors negatively impactHPV vaccine implementation in these populations, given itscurrent high cost.


3/13

296 Cancer Biology & Therapy Volume 11 Issue 3

are found in high-grade cervical dysplastic (also premalignant)lesions and in the majority of frankly invasive cervical cancers.Both HPV 16 and 18 are classed as high-risk oncogenic typesand are particularly likely to persist and progress from high-gradepremalignant cervical disease to invasive cancer. HPV types 31,33, 45, 52 and 58 are also recognized as oncogenic, but are lessprevalent in cervical cancer.3,4,6-9 The less prevalent oncogenic

types will not be covered in this discussion. HPV 16 and HPV18 are by far the most prevalent types in both symptomatic andasymptomatic women, and are associated with about 50–70%and 20–30% of invasive cervical cancers diagnosed worldwide,respectively.Table 1 shows the classication by risk type for thecommon genital HPV genotypes.

Four HPV 16 variants have been dened; European (E), African (Af-1, Af-2), Asian American (Aa). Similarly, three HPV18 variants are currently recognized; European (E), African (Af )and Asian Amerindian (AsAi).4,7 The existence of these variantsis generally thought to reect the migration and sexual behav-ioral patterns of human populations over time. Together, HPV16 and 18 cause over 95% of invasive squamous cell cervical can-cers diagnosed. Cervical adenocarcinomas are mostly associated with HPV 16, 18 and 45.4

Epidemiologic studies indicate that HPV genital-tropic typesare seen in both symptomatic and asymptomatic patients andthat the majority of infections spontaneously resolve. It is notuncommon in the clinical setting to have multiple HPV typespresent in the same patient. Multiple, simultaneous or sequentialgenital infections can occur, and in adolescent girls it is oftendifcult to distinguish between persistent infections and newinfections by different subtypes of HPV.11 While persistent infec-tion with an oncogenic HPV is necessary for carcinogenesis, itshould be stressed that several other factors and poorly dened

molecular events drive the transformation of cervical epithelialcells to a malignant state. Clinical studies have shown that suchtransformation is associated with integration of the viral genomeand epithelial cell genetic instability that occurs over a longperiod of HPV infection. The importance of genetic instabilityis highlighted by the increased risk of cervical cancer in patients with Fanconi’s anemia. Also signicant are immunosuppression,as seen in Human Immunodeciency Virus (HIV) co-infectionand chronic steroid therapy following organ transplantation, which increase the likelihood of persistence of an oncogenic HPVinfection and its sequelae.6,12 In fact, in HIV patients, cervicalcancer is a criterion for making a clinical diagnosis of AcquiredImmune Deciency Syndrome (AIDS).

Immunologic Aspects of HPV Infection

The majority of immunocompetent individuals infected withHPV are able to clear the viral infection and remain asymptom-atic. It is, however, controversial whether the virus is truly elimi-nated from the patient or suppressed to such an extent that it isundetectable with current sampling and analytic approaches. In aminority of patients, HPV infection persists and causes clinicallydetectable lesions that can progress to invasive cancer over a longtime period, typically measured in years to decades.9,13 It is not

HPV types and cervical disease. HPVs are small, non-enveloped DNA tumor viruses. Their genomes of approximately

8 kb are covalent closed circular, encoding eight genes, from theE1, E2, E4, E5, E6, E7, L1 and L2 open reading frames (ORFs).The genome is wrapped around nucleosomes and coated by a60 nm diameter T = 7 capsid. HPV is believed to have evolvedalong with humans over many eons and productively infects onlyhumans, although many other animal species of papillomavirusexist.7 Analyses of different HPV types have suggested a commonancestry, which has diversied over time to include host-specic‘types’ that have maintained a predilection for infecting basalepithelial cells of the skin or mucosal epithelia, notably of thegenitalia and oropharynx.7,8 In the 1970s, hybridization studiesusing DNA from skin and genital lesions suggested that there were differences in the HPV types found in biopsy specimens ofskin and genital lesions. Subsequent experiments reported in the1980s conrmed the existence of distinct HPV types in associa-tion with dysplastic genital warts and invasive cervical cancers.6,9 There are at least one hundred different types of HPV classi-ed phylogenetically within the alpha, beta, gamma, delta andmu genera based upon differences in their nucleotide sequences.‘Alpha’ genus HPV types infect the genital and oropharnygealmucosa exclusively and include the oncogenic HPV types associ-ated with cervical cancer. HPV ‘types’ are assigned on the basisof a difference of at least 10% in the nucleotide sequence of theL1 open reading frame. HPV ‘variants’ differ generally by


4/13


they are shed as part of the natural process of epithelial matu-ration. Traumatic micro-abrasions, such as occur during sexualintercourse, expose the naïve basal layer cells to HPV.14,15 Cellentry is not well understood but is believed to be receptor-mediated and multiple reports have implicated heparin sulfateas a candidate molecule involved in this process.16,17 HPV rep-lication is dependent upon and utilizes the normal replicativemachinery of the cervical cells, which is subverted by two viralproteins, E1 and E2. The virus is maintained at typically ~100episomal copies per basal cell and the initial infection triggers aburst of viral replication up to this level.

Basal cells that are infected with HPV continue to divide and

each form two daughter cells containing viral genomic material.One cell of the pair remains in the basal layer and retains itsdividing capacity, therefore acting as a repository for replicationof the virus, which requires active cell division to maintain itslife cycle. The other daughter cell continues upward through thesuprabasal layer, where it differentiates and eventually is shedfrom the epithelial surface.16,18 In order to ensure that cervicalcells are maintained in a state of constant growth and division,HPV early proteins are expressed, which stimulate and propagatecell growth via the actions of the E5, E6 and E7 genes. Uponcellular differentiation in the suprabasal layer, the viral genome is

entirely clear why HPV infection persists in certain individuals,but decits in cellular immunity, such as treatment of solid organtransplant patients with immunosuppressants and those co-infected with HIV, are associated with increased persistence andHPV related disease. Conversely, the prevention of new infec-tions by vaccination with L1 viral like particles (VLPs) appearsto be primarily affected by neutralizing antibodies. In animalmodels, passive transfer of serum immunoglobulins from L1VLP-immunized animals is sufcient to confer protection uponnaïve animals. Furthermore, the measurement of in vitro neu-tralization titers in sera is the best available correlate to assessprotection in patients for vaccine types and also cross-protection

against non-vaccine types, although no minimal titer for protec-tion has been dened as yet.

HPV Infection Cycle

As mentioned previously, HPV has a predilection for epithelialcells of the cervix, which are stratied into a non-differentiatedbasal monolayer and a suprabasal differentiated non-proliferatingepidermis. The basal layer sits above the basement membrane,below which is the cervical stromal layer. Dividing immaturebasal cells move upward through to the epidermal layer where

Figure 1. Low-grade lesion, which extends into endocervical canal.


5/13


cellular DNA replication factors. By binding to sites that areproximal to its promoter, E2 leads to the recruitment of E1 tothe HPV origin of replication. E1 binding to E2 increases stablebinding to the AT-rich sequences within the origin of replicationthrough the formation of a binding complex.

E2 contains about 360 amino acids and possesses a DNAbinding domain as well as a transactivation domain, and itcan be spliced into a truncated variant lacking the transactiva-tion domain. Thus, through alternative splicing, E2 is capableof both activation and repression of its target proteins in HPV.Interestingly, E2 also downregulates E6 and E7. Since E2 expres-

sion is typically lost during viral integration and carcinogenicprogression, E6 and E7 are relieved of the suppressive inuenceof E2, leading to elevated expression of these viral oncogenes.18,19

By repression of an early promoter, E2 self-regulates levels ofE1 and E2 transcription to maintain a stable viral copy number(~102/cell) in the basal epithelia. As keratinocyte differentiationprogresses in the upper layers of squamous epithelia, the tran-scription of E1 and E2 switches to a different (late) promoter thatis not modulated by E2, thus allowing for vegetative replicationand increased (~104/cell) viral genomic copies. Following this,expression of the capsid antigens leads to the formation of

replicated to 10,000 or greater copies/cell, and expression of thelate viral genes E4, L1 and L2 is triggered. The L1 (major) and L2(minor) proteins form the capsid structure around the genomicmaterial of the virus. Once this assembly is complete within thecells, the mature viral particles are released from the epithelialcells during terminal shedding from the epithelial surface.16,18,19 It is postulated that the E4 viral protein facilitates the release andspread of HPV from the keratin cage within keratinocytes by col-lapsing keratin laments in the dying squames.

It should be noted that carcinogenic progression is not partof the normal HPV life cycle, but rather a non-productive ‘dead

end’ that is only associated with a small subset of virus types andoccurs only after a long period of infection. The vast majority ofinfections are benign and self-limited.

HPV Virology

E1 and E2. The initial events after HPV achieves cell entryand delivery of the genome to the nucleus trigger the expressionof the HPV early genes E1 and E2. The E1 and E2 genes acti-vate viral replication through interaction with specic sequencesin the HPV genome origin of replication,18,19 and by co-opting

Figure 2. High-grade lesion with features of vascular punctation and mosaic pattern.


6/13


downregulation, suggesting a role for E5 in viral escape fromcellular immunity.

E6. The HPV E6 oncoprotein is a 150 amino acid zinc-binding protein that does not possess enzymatic activity. E6 con-tains two zinc nger motifs, which possess four C-X-X-C motifsnecessary for E6 function.21 The E6 oncoprotein induces cellular

transformation, however, on its own, it has weak transformingproperties, but serves to amplify the transforming effect of HPVE7 oncoprotein, which will be discussed later. E6 interacts pri-marily with the p53 tumor suppressor protein,22-24 as well as withother cell cycle regulators like Bak, Blk and cell adhesion pro-teins. It is known that the E6 oncoprotein of low-risk HPV typesis less potentiating than that of high risk HPV types with respectto cell transformation and immortalization activity.25

The E6AP is an E3 ubiquitin ligase that is responsible forthe targeted proteasomal degradation of cellular proteins. TheLXXLL domain on E6AP has been characterized as the binding

viral particles by encapsidation of the nucleosome-bound viralgenomes.

E5. As part of the early replication process, the E5 geneis expressed. E5 is a small, hydrophobic, single membrane-spanning protein that complexes with platelet derived growthfactor receptor and epidermal growth factor receptor to stimu-

late cell growth.19

E5 also inhibits apoptosis, and maintains theepithelial cell in continuous replication. Much of the specicactivities and function of E5 are poorly understood, however,it appears that this gene is involved mainly in early events andhas an unknown role in the later phases of viral replication. Although it has oncogenic activity, E5 expression is usuallyabsent in malignant cervical cells,20 and this has led to sugges-tions that E5 activity is not critical for epithelial t ransformation.E5 also binds to the vacuolar H+ ATPase and inhibits its abilityto lower endosomal pH, potentially impacting vesicular traf-cking. Indeed, E5 expression is also associated with MHC-I

Figure 3. High-grade lesion with punctation and mosaicism also noted.


7/13


p53 and preventing the activation of downstream pro-apoptoticgenes. Continued E6-E6AP binding leads to the targeting of theBlk protein for breakdown, with the resultant effect of uncheckedcell proliferation.27

E6 also induces cell proliferation by targeting the expressionof apoptotic proteins other than p53. E6 binding leads to thedegradation of Bak, a Bcl-2 family pro-apoptotic protein that iscrucial in mitochondrial pore formation during initiation of the

intrinsic apoptotic cascade. Bak degradation prevents the releaseof cytochrome c from the mitochondria, and prevents the down-stream activation of caspases necessary for apoptosis.27,28 E6 ofhigh-risk HPV type has also been shown to disrupt the extrinsicapoptotic pathway by binding to TNFR-1 (tumor necrosis factorreceptor), Fas, CD95 ligand and TNF related apoptosis induc-ing ligand (TRAIL) receptors,28 thus inhibiting the downstreamrelease of caspase 8 and caspase 10, as well as inhibiting the for-mation of the death inducing signaling complex (DISC).

As mentioned above, the cervical basal (epithelial) cells rest onthe basement membrane, glued to the extracellular matrix (ECM).

site of E6 proteins of the alpha papillomaviruses. High risk HPVE6 oncoprotein rst binds to E6AP, before it is able to bind p53to form a trimer. Within this trimeric complex, E6AP targetsp53 for disposal by proteosomes in the cervical epithelial cellvia ubiquitination.21 Under normal cell conditions, p53 exists inlow levels, tightly regulated by the E3 ubiquitin ligase activity ofMdm2. The low level of p53 in normal epithelial cells is upregu-lated via inhibition of Mdm2 when DNA damage occurs or when

abnormal cell proliferation signals are detected.22

In response tothe upregulation of p53 activity, several anti-proliferation path- ways are activated. One of these is the activation of genes via theaction of novel proteins such as p300.22,26 The loss of p53, whichis a key regulator of uncontrolled cell proliferation, ensures thatthe cervical epithelial cell is unable to exit the cell cycle. Highrisk HPV E6 binds to the core region of p53, thereby inducingp53 degradation, which occurs when the core region is bound.This ability to degrade p53 is unique to high-risk HPV types, asthe low risk types do not bind to the core region. Also, high-riskHPV E6 has been shown to bind p300, causing deacetylation of

Figure 4. Low-grade lesion with skip pattern.


8/13


deregulates proliferation and disrupts the cell-cell junctions viadisruption of signals that regulate cell-cell contact. PDZ bindingappears to be an interaction that is necessary for carcinogenesis,since E6 oncoproteins isolated from almost all invasive cervicalcancers possess this highly conserved PDZ binding motif.23,28,30 E6 in low risk HPV types have not been shown to possess thisPDZ binding motif.

Tissue culture studies have suggested a key mechanism by

which E6 immortalizes epithelial cells through the stimulationof telomerase activity. High-risk HPV type E6 has been shownto activate telomerase through E6AP binding to activate hTERT(human telomerase reverse transcriptase). This process is alsodependent on the c-Myc oncogene, which acts on hTERT pro-moter to increase its transcription. In addition, it appears that invivo, E6-E6AP binding increases hTERT activity through inter-action with two isoforms of a gene repressor, NFX-1 (nuclear fac-tor binds to the X1 box). The ubiquitination of NFX1-91 preventsrepression of the hTERT promoter, while binding to NFX1-123leads to direct induction of hTERT promoter activity.22

Cell signals from the ECM, which travel through the actin cyto-skeleton of the basement membrane to the cell nucleus, play a rolein basal cell growth; once basal cells begin their ascent throughto the epithelial surface, the growth signals stop and epithelialdifferentiation dominates. This transition is tightly regulated anddisruption of this process is one of the mechanisms by which E6transforms cells. The anchoring of the basal cells to the ECMinvolves paxillin and zyxin adhesion molecules. Their binding by

E6 leads to disruption of normal cell attachment and cell signal-ing. In concert, the detachment of the epithelial cells from theextracellular matrix, as well as the prevention of terminal dif-ferentiation, serve to maintain an environment of continued viralreplication within continuously dividing epithelial cells.

The E6 protein of high-risk HPV types possess a PDZ-bindingmotif that is critical for cell transformation through the bindingof cell proteins that have PDZ domains. In vitro studies haveidentied these proteins such as hScribble and hDLG (tumorsuppressors), MUPP1 and PATJ (tight junction proteins).22,29 The proteosomal degradation of these proteins by E6 binding

Figure 5. Invasive cervical cancer in a 23 year old patient with HIV infection.


9/13


that E7 inhibition of CKIs p15, p21 and p27 is partly responsiblefor the absence of effects of TGFbeta in infected epithelial cells.29

E6 modulates the contact between epithelial cells and antigenpresenting cells (Langerhans cells) in the epidermis by af fectingthe cell surface protein, E-cadherin. Diminished Langerhans cellnumbers and reduced E-cadherin are recognized features of HPVinfection. Epithelial cells normally secrete cytokines to induce

chemotaxis, which leads to the repopulation of Langerhan’s cellprecursors.27 This chemotactic activity is mediated through themacrophage inammatory protein, MIP-3alpha. MIP-3alphaexpression is inhibited in cells that show evidence of E6 and E7expression with the resultant impairment of antigen presentation.

In addition to the above E6 and E7-mediated immune effects,the HPV replication cycle appears to confer certain survivaladvantages that allow for HPV infections to persist for long peri-ods in certain individuals. The intraepithelial sequestration of thereplication cycle allows for the absence of a blood borne phase.23 This absence of viremia allows viral replication to proceed some- what covertly without triggering the immune system. The releaseof viral particles from the terminal (apical or top layer) epithe-lial cells, as well as non-lytic replication, avoids provocation ofanti-inammatory molecules. Asymptomatic HPV infections arethe norm in immunocompetent women, even in those with high-risk HPV infections, because most (younger) women clear theirinfections within 12 months.35 This observation is obviously con-founded by the fact that it is quite possible for the same patientto acquire different HPV types going forward in time, such thatthe contribution of one or an other HPV type to the clinical pic-ture of persistence is hard to separate. The reason for persistenceand progression of lesions in a minority of patients is not wellunderstood. The average lag time from initial HPV infection tocarcinogenesis is 10–20 years.35 The integration of viral DNA

into host cells is a typical event in carcinogenesis, but is thoughtto occur sporadically. This suggests that there may be other, yetunknown, environmental and genetic factors that inuence thepropensity of HPV to remain latent, regress or progress with time.

Clinical Aspects of HPV Infection—Premalignant Cervical Disease

The cervix normally contains a central canal (endocervical canal), which is the caudad continuation of the endometrial canal of theuterus. Columnar cells and squamous cells line the proximal anddistal endocervical canal, respectively.36 The squamo-columnarcellular junction is an active transition zone known as the ‘trans-

formation zone’ (TZ), which is the site of origin of the majorityof cervical dysplastic (premalignant) lesions and carcinomatouslesions. In premenarcheal and postmenopausal women, hor-monal and physical changes cause the transformation zone torecede into the cervical canal, hence making it sometimes dif-cult to visualize on vaginal examination. In reproductive age women, the acid environment of the vagina leads to destructionof the columnar cells of the TZ, and induces ‘squamous metapla-sia’ in the TZ. This metaplastic change (clinically considered asbenign) occurs when the ‘reserve cells’ underlying the columnarepithelium differentiate into a squamous type epithelium. HPV

E7. E7 contains about 100 amino acids and, like E6, lacksintrinsic enzymatic activity.31,32 E7 exerts its oncogenic effectprimarily by binding with retinoblastoma protein (pRb) andthe related pocket proteins, p107 and p130. By modulating E2Ftranscriptional factors, these proteins regulate cell proliferationby controlling entry at the G1/S phase of the cell cycle. Bothlow risk and high risk HPV E7 bind to pRb, however, the low

risk HPV E7 has much lower afnity due to a single amino acidsubstitution in the binding domain. E7 binding to pRb resultsin proteosomal degradation of pRb and the unrestricted tran-scriptional activity of E2F.31-33 Direct binding of E7 to E2F alsoensures transcriptional activity that maintains the epithelial cellso they are always ready to enter S phase. E2F regulates the Sphase of the cell cycle by control of cyclins, specically cyclins A and E. Through the pRb inhibition and direct E2F inductionmentioned above, E7 drives cell proliferation with increased lev-els of cyclins and also inhibits the activities of cyclin-dependentkinase inhibitors (CKI), p21 and p27.33 E7 binding to pRb is alsolinked to the expression of c-jun, which is important in control-ling cell differentiation and growth. In effect, the inhibition ofpRb and c-jun ensure that the cell is maintained in a state of Sphase progression.34

Similarly, E7 binds to histone deacetylases (HDACs) withthe resultant effect of increasing E2F-driven transcription andS phase progression in epithelial cells. In addition to cell cycledysregulation, E7 mediates various structural changes in thegenomic structure of epithelial cells, notably extra chromosomalmaterial, polar mitoses, anaphase bridging and aneuploidy. Forexample, E7 uncouples centriole synthesis from cell cycle con-trol, resulting in aberrant numbers of centrioles and chromosomemissegregation. The activation of cdk2 by E7 is thought to bepartly responsible for these changes,34 and in vitro experiments

have demonstrated that cdk2 inhibition is associated with adecreased frequency of these chromosomal changes.

E6 and E7 Immune Modulation

In addition to the combined expression of E6 and E7 in high-riskHPV types greatly enhancing cellular transformation, E6 andE7 also modulate molecules involved in the innate and adaptiveimmune responses, potentially enabling HPV to evade immunesurveillance, especially in the early stages of replication.

E6 inhibits the ability of interferon regulatory factor 3 toinduce the activation of interferon beta, thus abrogating this earlyresponse of the innate immune system to viral infection. E7 also

binds to interferon regulating factor 1 to prevent activation ofinterferons alpha and beta. Likewise, the inhibition of toll likereceptor 9 (TLR9) and subsequent failure of cytokine productionby the expression of E6 and E7 has been demonstrated in tissueculture studies.29

Cervical cancer cells show an abnormal response to the actionof transforming growth factors (TGFs), which typically act toprevent uncontrolled epithelial cell growth. Downregulation oftumor necrosis factor (TNF)alpha expression, as well as resis-tance to the effects of TNF alpha and beta, have been observed incervical cancer cells. While not entirely understood, it is thought


10/13


as mild, but which may not show overt features of a high-gradeprocess. From a clinical perspective, one of the most challengingdilemmas has been the issue of avoiding overtreatment of inde-terminate and low-grade lesions, while trying to devise clinicalthresholds to capture lesions that have a reasonably high chanceof progressing to cancer.Table 2 shows regression, persistenceand progression rates for dysplastic lesions. Please seeTable 3 .The management of these lesions is discussed below.

Clinical Aspects of HPV Infection—Management Strategies

Cervical Pap smear screening in women in the US is instructedto commence three years after initiation of sexual activity, andno later than age 21 years. Of the 50 million cervical Pap smearsdone annually in the US, about 3–4% are reported as LSIL,0.6% as HSIL and 4–6% as ASCUS.35 In recent years, high-risktype HPV testing has been used to guide the management ofabnormal cervical Pap smears. The ASCUS/LSIL Triage Study(ALTS), carried out from 1995–98, was reported as a random-ized trial of 3,488 women with ASCUS Pap smears and 1,572 women with LSIL Pap smears, using HPV testing to determine

colposcopy referral and setting HSIL as the threshold lesion forcolposcopy referral (study endpoint).40,41 Women were random-ized to three groups; immediate colposcopy (IC), HPV testingand conservative management (CM) groups.

In the LSIL part of the study (n 1,572), a breakdown of theenrollment Pap smears on re-examinations were as follows; nega-tive 18.7%, LSIL 45%, ASCUS 23.2%, HSIL (CIN 2) 11.3%and HSIL (CIN 3) 1.4%. Using the triage strategies of IC,HPV test and CM, the sensitivities for detecting CIN 3 overtwo years follow up (study endpoint) were 55.9, 65.9 and 48.4%respectively (p 0.16). Based on these detection sensitivities, the

infection can induce change from normal to dysplastic cellulararchitecture (clinically considered as abnormal) in the transfor-mation zone and surrounding cervical portio.

The old terminology for cervical dysplasia was ‘CervicalIntra-epithelial Neoplasia’ or ‘CIN’, which has since changedto ‘Squamous Intraepithelial Lesion’ (SIL) following the 2001revision of the Bethesda Classication of cervical dysplasia.36 Cervical cancer screening involves the initial step of accessingsquamous cells in the transformation zone, for cytological analy-sis (Papanicolau smear or Pap smear). Cervical cytology reportsclassify dysplastic lesions as low grade or high-grade squamousintraepithelial lesions (LSIL, HSIL respectively), as well as atypi-cal squamous cells—‘unknown signicance’, or ‘cannot excludehigh-grade’ (ASC-US, ASC-H respectively). Cervical glandularcell abnormalities may also be reported, but will not be covered inthis discussion. If indicated, the next step following a Pap smearis the assessment of the cervix using a microscope (‘colposcopy’).Colposcopy usually facilitates the obtaining of biopsies fromthe cervical lip and endocervical canal. Histological reports ofcolposcopic biopsy specimens are reported as CIN 1, 2 or 3. In2001, ‘LSIL’ and ‘HSIL’ were introduced in the Bethesda revisioncolposcopic biopsy descriptions; however, ‘CIN’ is still used inmany biopsy reports.36

LSIL typically involves the basal one-third of the cervicalepithelial layer (above the basement membrane and stroma),

and occurs as a result of HPV cytopathic effect of a productivelesion, i.e., the complete virus life cycle. LSIL lesions displaya pathognomic change known as ‘koilocytosis’.37 Koilocytosisrefers to the presence of nuclear atypia in combination withcytoplasmic cavitations, which gives a vacuolated appearanceto the cells. The cytoplasm takes on a granulated and coarseappearance under the microscope. In addition, abnormali-ties in the chromosomal structures involved in mitosis createa multinucleated appearance of the epithelial cells. Presence ofimmature basaloid cells in the upper third of the cervical epithe-lial layer characterizes HSIL. Affected cells show cytoplasmicgranulation, nuclear crowding, increased mitotic activity, cel-lular crowding (loss of polarity), as well as variations in the size

of their nuclei (anisonucleosis).38

There may also be an increasein the nuclear to cytoplasmic size ratio due to shrinkage of thecytoplasm. Under the old terminology, HSIL corresponds toCIN 2 and CIN 3. The most severe HSIL change (formerlytermed carcinoma-in situ, CIS) involves the whole thickness ofthe cervical epithelium, however, an important distinguishingfeature from invasive carcinoma is the absence of disruption ofthe basal membrane of the epithelial layer and lack of invasioninto the cervical stroma.39 ASCUS lesions denote a group of dys-plastic lesions that are not clearly LSIL or HSIL ; in other words,lesions that the pathologists do not feel comfortable describing

Table 2. Rates of regression and progression of cervical dysplasialesions 36

Regress % Persist % Progress %

CIN 1/2 57 27 15

CIN 3 8 69 22

Table 3. HPV genes and their functions

HPV gene Function

E1, E2

Viral replication

Autoregulation of E1 and E2 function by E2

Repression of E6 and E7 expression by E2

E5Inhibition of apoptosis

? Immune dysregulation

E6

p53 degradation

Inhibition of apoptosis

Cell cycle progression

Cell transformation

Immune dysregulation

E7

pRb inhibition

Cell cycle progression

Cell transformation

Immune dysregulation

E4 ? Facilitates release of mature viral particles

L1, L2Synthesis of viral capsid

Antigenicity


11/13


colposcopy referrals, and this is troublesome for a low detectionrate and higher chance of missing high-grade persistent lesions.

Based in part on clinical data reviewed above,40-42 the American College of Obstetricians and Gynecologists (ACOG),and the American Society for Colposcopy and Cervical Pathology(ASCCP) have endorsed revisions to cervical Pap smear screeningand management as follows:

The time interval for Pap smear testing of women consideredto be at low risk (using risk factor screening and prior Pap smearresults) has been extended from one- to two-yearly. Women overthe age of 30 years who have negative HPV tests, preceded by atleast two consecutive negative Pap smears can be screened onceevery three years. Among women in this group, the 10 year risk ofCIN 2 is 2–5%, and of CIN 3 is


12/13


Conclusion

The study of HPV has provided valuable information on themechanisms by which tumor viruses initiate carcinogenesisand the regulation of cell cycle in human cells. Cervical canceras a disease model presents unique opportunities in nding acure, because it is caused by a pathogen with well character-

ized biological mechanisms and life cycle. The use of cytologicand HPV screening and ablation of premalignant disease hasproven very successful at reducing cancer rates in developednations. The recent introduction of prophylactic HPV vac-cines has been another great step forward. Given the huge dis-ease burden in the developing parts of the world, it is evidentthat population based measures offer the best hope of limitingmortality worldwide. The development of therapeutic cervicalcancer vaccines and/or virus-targeted drug therapies is eagerlyawaited.

Acknowledgements

Dr. Ibeanu wishes to acknowledge Dr. Richard B. Roden forhis critical review of this manuscript.

cervical dysplastic lesions in women aged 16–26 years, who were not infected by any of the vaccine HPV types.47 Efcacyin women with simultaneous vaccine type HPV DNA positivityand seropositivity was about 25%. The most common adverseeffects were related to injection site pain (84 vs. 48.6% in treat-ment and placebo groups respectively). It is expected that thevaccines will achieve a lifetime risk reduction of 20–70% for

cervical cancer, in patients aged 12 years. The vaccine is typi-cally given in three doses of 0.5 ml at 0, 2 and 6 months. Thebivalent vaccine contains recombinant L1 protein from HPV 16and 18, and phase III study of 18, 644 females followed for 35months showed efcacy of up to 93% in prevention of CIN 2lesions due to HPV 16 and 18.46 Similar to the quadrivalentvaccine, there was a higher rate of complications at the injectionsite. The dosage schedule is 0.5 ml at 0, 1–2, and 6 months fora total of three doses. New approaches to vaccine developmentmay provide more options for controlling HPV acquisition andtransmission in the future. The use of recombinant L2-basedprophylactic vaccines (Roden R, unpublished data), as well asthe development of drug targets against early protein (E6 andE7) activity are a few examples.

References1. Parkin D. The global health burden of infection-

associated cancers in the year 2002. Int J Cancer 2006;118:3030-44.

2. McLaughlin-Drubin M, Munger K. Viruses associ-ated with human cancer. Biochim Biophys Acta 2008;1782:127-50.

3. Chauhan S, Jaggi M, Bell M, Verma M, Kumar D.Epidemiology of human papillomavirus in cervicalmucosa. Methods Mol Bio 2009; 471:439-56.

4. Einstein M, Schiller J, Viscidi R, Strickler H, CoursagetP, Tan T, et al. Clinician’s guide to human papillomavi-rus immunology: knowns and unknowns. Lancet 2009;9:347-56.

5. American Cancer Society. Cancer facts and figures. Atlanta: American Cancer Society 2008.6. zur Hausen H. Papillomaviruses in the causation of

human cancers—a brief historical account. Virol 2009;384:260-5.

7. Lizano M, Berumen J, Garcia-Carranca A. HPV-relatedcarcinogenesis: Basic concepts, viral types and variants.

Arch Med Res 2009; 40:428-34.8. De Villiers E, Fauquet C, Broker T, Bernard H, Zur

Hausen H. Classification of papillomaviruses. Virol2004; 324:17-27.

9. zur Hausen H. Papillomaviruses and cancer: from basicstudies to clinical application. Nat Rev 2002; 2:342-50.

10. Schiffman M, Rodriguez A, Chen Z, Wacholder S,Herrero R, Hildesheim A, et al. A population-basedprospective study of carcinogenic human papilloma-virus variant lineages, viral persistence and cervicalneoplasia. Cancer Res 2010; 70:3159-69.

11. Ho G, Bierman R, Beardsley L, Chang C, Burk R.Natural history of cervicovaginal papillomavirus infec-tion in young women. N Engl J Med 1998; 338:423-8.

12. Lehoux M, D’Abramo C, Archambault J. Molecularmechanisms of human papillomavirus-induced carci-nogenesis. Pub Health Genomics 2009; 12:268-80.

13. Wang S, Hildesheim A. Viral and host factors in humanpapillomavirus persistence and progression. J NatCancer Inst Monogr 2003; 31:35-40.

14. Doorbar J. Molecular biology of human papillomavirusinfection and cervical cancer. Clin Sci 2006; 110:525-41.

15. Hebner C, Laimins L. Human papillomaviruses: Basicmechanisms of pathogenesis and oncogenicity. RevMed Virol 2006; 16:83-97.

16. Letian T, Tianyu Z. Cellular receptor binding and entryof human papillomavirus. Virol J 2010; 7:1-7.

17. Giroglou T, Florin L, Schafer F, Streeck R, Sapp M.Human papillomavirus infection requires cell surfaceheparan sulfate. J Virol 2001; 75:1565-70.

18. Hamid N, Brown C, Gaston K. The regulation of cellproliferation by the papillomavirus early proteins. CellMol Life Sci 2009; 66:1700-17.

19. Stubenrauch F, Laiminis L. Human papillomavirus lifecycle: active and latent phases. Sem Cancer Biol 1999;9:379-86.

20. Cullen A, Reid R, Campion M, Lörincz AT. Analysisof the physical state of different human papillomavirusRNAs in intraepithelial and invasive cervical neoplasia.

J Virol 1991; 65:606-12.

21. Schwarz E, Freese U, Gissman L, Mayer W, RoggenbuckB, Stremlau A, et al. Structure and transcription ofhuman papillomavirus sequences in cervical carcinomacells. Nature 1985; 314:111-4.

22. Howie H, Katzenellenbogen R, Galloway D.Papillomavirus E6 proteins. Virol 2009; 384:324-34.

23. Yugawa T, Kiyono T. Molecular mechanisms of cervicalcarcinogenesis by high-risk human papillomaviruses:novel functions of E6 and E7 oncoproteins. Rev MedVirol 2009; 19:97-113.

24. Wise-Draper T, Wells S. Papillomavirus E6 and E7proteins and their cellular targets. Front Biosci 2008;13:1003-17.

25. Yim E, Park J. The role of HPV E6 and E7 oncopro-teins in HPV-associated cervical carcinogenesis. CancerRes Treat 2005; 37:319-24.

26. Munger K, Baldwin A, Edwards K, Hayakawa H,

Nguyen C, Owens M, et al. Mechanisms of humanpapillomavirus-induced oncogenesis. J Virol 2004;78:11451-60.

27. Ghittoni R, Accardi R, Hasan U, Gheit T, Sylla B,Tomassino M. The biological properties of E6 andE7 oncoproteins from human papillomaviruses. VirusGene 2010; 40:1-13.

28. Scheffner M, Huibregtse J, Vierstra R, Howley PM.The HPV-16 E6 and E6-AP complex functions as aubiquitin-protein ligase in the ubiquitination of p53.Cell 1993; 75:495-505.

29. Martinez-Lagunas A, Madrid-Marina V, Gariglio P.Modulation of apoptosis by early human papillomavi-rus proteins in cervical cancer. Biochim Biophys Acta2010; 1805:6-16.

30. Thomas M, Narayan N, Pim D, Tomaic V, MassimiP, Ngasaka K, et al. Human papillomaviruses, cervicalcancer and cell polarity. Oncogene 2008; 27:7018-30.

31. McLaughlin-Drubin M, Munger K. The human papil-lomavirus E7 oncoprotein. Virol 2009; 384:335-44.

32. Liu X, Clements A, Zhao K, Marmorstein R. Structureof the human papillomavirus E7 oncoprotein andits mechanism for inactivation of the retinoblastomatumor suppressor. J Biol Chem 2006; 281:578-86.

33. Ganguly N, Parihar S. Human papillomavirus E6 andE7 oncoproteins as risk factors for tumorigenesis. JBiosci 2009; 34:113-23.

34. Mammas I, Sourvinos G, Giannoudis A, SpandidosD. Human papillomavirus (HPV) and host cellularinteractions. Pathol Oncol Res 2008; 14:345-54.

35. zu r Hausen H. Papillomaviruses causing cancer:Evasion from host-cell control in early events in carci-nogenesis. J Nat Cancer Inst 2000; 92:690-8.

36. Wright TC, Kurman RJ, Ferenczy A. Precancerouslesions of the cervix. In: Kurman RJ, (Ed). Blaustein’sPathology of the Female Genital Tract, 5th Ed. Springer,New York 2002; 253-324.

37. Wright T, Kurman R. A critical review of the mor-phologic classification systems of preinvasive lesionsof the cervix: the scientific basis of the paradigm.Papillomavirus Rep 1994; 5:175-81.

38. Fu Y, Braun L, Shah K, Lawrence WD, Robboy SJ.Histologic, nuclear DNA and human papillomavirusstudies of cervical condylomas. Cancer 1983; 52:1705-11.

39. Holowaty P, Miller A, Rohan T, To T. Natural history ofdysplasia of the uterine cervix. J Natl Cancer Inst 1999;91:252-8.

40. The ASCUS-LSIL Triage Study (ALTS) Group. Resultsof a randomized trial on the management of cytol-ogy interpretations of atypical squamous cells of unde-termined significance. Am J Obstet Gynecol 2003;188:1383-92.

41. The ASCUS-LSIL Triage Study (ALTS) Group. Arandomized trial on the management of low-gradesquamous intraepithelial lesion cytology interpreta-tions. Am J Obstet Gynecol 2003; 188:1393-400.

42. Castle P, Schiffman M, Wheeler C, Solomon D.Evidence for frequent regression of cervical intraepithelialneoplasia—grade 2. Obstet Gynecol 2009; 113:18-25.

43. Hildesheim A, Schiffman M, Gravitt P, Glass A, GreerC, Zhang T, et al. Persistence of type-specific humanpapillomavirus infection among cytologically normal

women. J Infect Dis 1994; 169:235-40.


13/13

47. Garland S, Hernandez-Avila M, Wheeler C, Perez G,Harper D, Leodolter S, et al. Quadrivalent vaccineagainst human papillomavirus to prevent anogenitaldiseases. N Engl J Med 2007; 356:1928-43.

46. Paavonen J, Naud P, Salmeron J, Wheeler C, ChowS, Apter D, et al. Efficacy of human papillomavirus A(HPV)-16/18 ASO4-adjuvanted vaccine against cervi-cal infection and precancer caused by oncogenic HPVtypes (PATRICIA); final analysis of a double-blind,randomized study on young women. Lancet 2009;374:301-14.

44. Evander M, Edlund K, Gustafsson A, Jonsson M,Karlsson R, Rylander E, et al. Human papillomavirusinfection is transient in young women: a population-based cohort study. J Infect Dis 1995; 171:1026-30.

45. Markowitz L, Dunne E, Saraiya M, Lawson H, ChessonH, Unger E. Quadrivalent human papillomavirus vac-cine: recommendations of the Advisory Committee onImmunization Practices (ACIP). MMWR RecommRep 2007; 23:1-24.

molecular pathogenesis of cervical cancer

Documents