Human papillomavirus and molecular considerations for cancer risk

Abstract

Human papillomaviruses (HPVs) are a major cause of cancer globally, including cervical cancer. The HPV ‘early’ proteins, E6 and E7, are the chief oncoproteins involved in cancer progression. These oncoproteins are more highly expressed in high-grade dysplasias and invasive cancer coincident with reduced viral DNA replication and reduced production of infective progeny virions. The E6 and E7 oncoproteins interact with several cellular proteins—classically TP53 and RB1, respectively—leading to the degradation of several of these proteins, although all interactions do not necessarily result in the degradation of a cellular protein. HPV infection is also associated with viral and host DNA methylation changes, many of which also occur in cancer types not associated with HPV infection. The E6 and E7 interactions with cellular proteins and DNA methylation changes are associated with changes in the integrity of key cellular pathways that regulate genomic integrity, cell adhesion, the immune response, apoptosis, and cell cycle control. The alterations in key cellular pathways may provide useful biomarkers to improve the sensitivity of current cancer screening methods, such as the Papanicolaou test. This review provides a detailed summary of the interactions of E6 and E7 with cellular proteins and alterations in cellular DNA methylation associated with HPV infection. The importance of molecular biomarkers to the clinical setting, underserved populations, and general public health is discussed. Cancer 2008;113(10 suppl):2981–94. Published 2008 by the American Cancer Society.

Human papillomaviruses (HPVs) are a major cause of cancer morbidity and mortality. HPV is classified as a necessary, but insufficient, cause of cervical cancer. Although the relation between HPV and most epithelial cancers is unknown, HPV contributes to the development of oral cavity, oropharyngeal, penile, anal, vulvar, and vaginal cancers. The molecular mechanisms of HPV and its association with cancer risk have been most thoroughly studied in cervical tissue and cell lines, and these studies form the basis of our current knowledge. The paradox is that infection with oncogenic types of HPV is very common and most of these infections go unnoticed. Malignancy is a very rare outcome of a common infection, and it clearly involves complex interactions of the host, the environment, and the virus. The purpose of this article is to provide a broad overview of how HPV biology relates to oncogenesis and to summarize recognized molecular and epigenetic interactions of the host and virus that are believed to contribute to the molecular pathogenesis of HPV-associated cancers. The public health benefit of understanding this information is that it may be used to interpret epidemiologic data suggesting a causal role for HPV in cancer, as well as to suggest avenues for exploring biomarkers that can be used to improve screening and monitor therapeutic interventions.

HPV Biology

HPV are nonenveloped viruses composed of a double-stranded, closed circular (episomal) DNA genome, approximately 8 kilobases (kb) in size. The genome is divided into 3 regions: an upstream regulatory region (URR) or long control region (LCR) that regulates transcription and replication of the viral genome; the ‘early’ region, generally, with some exceptions,1 composed of 6 open reading frames (ORFs) labeled E1, E2, E4, E5, E6, and E7; and a ‘late’ region with 2 ORFs coding for viral structural proteins L1 and L2, the major and minor capsid proteins, respectively. L1 assembles into virions or viral-like particles, and it presents conformational epitopes recognized by neutralizing antibodies. L2 functions in viral capsid assembly and encapsulation in the nucleus of the host cell. The E6 and E7 proteins of oncogenic HPV are generally recognized as the dominant oncoproteins.2 HPV E2 is an important ‘early’ protein that transcriptionally represses E6 and E7, and loss of E2 during viral integration may contribute to oncogenesis as a result of unregulated transcription of E6/E7.

Viral Life Cycle

HPV infection is limited to epithelial cells, and completion of the viral life cycle requires epithelial differentiation.3, 4 A stratified squamous epithelium such as the cervix includes a basal/parabasal layer, adjacent to the basement membrane that is responsible for replenishing the superficial layers; the midzone, in which the epithelial cells mature and differentiate; and, lastly, the superficial layer, comprised of endstage fully differentiated cells. HPV infects basal cells that are accessible either through abrasions or at a junction in which the epithelium changes from stratified squamous to a columnar (glandular) type. In the cervix, this squamocolumnar junction occurs in the general region of the cervical os and the columnar (glandular) epithelium changes to squamous metaplasia, resulting in the transformation zone.

In basal cells, the viral DNA is usually maintained at low copy number in an episomal (circular) form. Viral replication is dependent on cellular machinery and transcription is dependent on cellular transcription factors expressed in differentiating cells. Because differentiating cells normally exit the cell cycle, a key role of E6 and E7 expression is to maintain active DNA replication in these differentiating cells via inactivation of p53 (TP53) and retinoblastoma protein (RB1). The viral DNA is replicated to high copy numbers in the midzone and encapsulated toward the surface. Viral progeny are released from superficial cells as they are shed, allowing another infective cycle to begin without lysis or destruction of viable cells. In cervical mucosa, productive HPV infection appears as cervical intraepithelial neoplasia grade 1 (CIN 1), with characteristic koilocytes. The majority of productive HPV infections are cleared or suppressed by cell-mediated immunity, resulting in regression of CIN I to a normal-appearing epithelium.

Molecular Basis for Viral-induced Oncogenesis

Oncogenesis can be viewed as an abortive viral infection, in which the virus persists in its cellular host and does not complete the normal life cycle.5 Infections that persist over time are more likely to progress to CIN 2/3. The molecular events underlying HPV persistence and progression to CIN 2/3 are not fully understood.6 Although E6/E7-mediated degradation of TP53 and RB1 allows viral replication, it also increases genomic instability. Loss of differentiation affects coordinated viral protein expression. As the viral host interaction shifts from infection to preneoplasia (CIN 2/3) and cancer, viral DNA may integrate into the host cell genome,7 further contributing to E6 and E7 overexpression. E6 and E7 also interact with several other important cellular proteins (Table 1) affecting the integrity of key pathways, as demonstrated in several in vitro studies.

Table 1. HPV E6 and E7 Binding to Cellular Proteins

HPV Protein	HUGO Symbol	Cellular Binding Partner	Binding Partner Function
Cellular Process*
HPV indicates human papillomavirus; HUGO, Human Genome Organisations; APC, adenomatous polyposis coli; kD, kilodalton. * The majority of genes contribute to several cellular processes. † The effect of E7 on CDKN2A is indirect.
Adhesion
E6	DLG458	Discs, large homolog 4 (Drosophila)	Cell-cell adhesion; membrane-associated scaffold protein
	DLG159	Discs, large homolog 1(Drosophila)	Interacts with APC; membrane-associated scaffold protein
	SCRIB60	Scribbled homolog (Drosophila)	Membrane-associated scaffold protein; localized to tight junctions; interacts with APC
	INADL61	InaD-like (Drosophila)	Maintenance of cell polarity by regulating formation of tight junctions
	PXN62	Paxillin	Multidomain adaptor protein; integrin signaling; cell motility and adhesion
	ZYX63	Zyxin	Actin-binding protein
	MAGI164, 65	Membrane-associated guanylate kinase, WW and PDZ domain containing 1 (MAGI proteins)	Localized to cellular tight junctions
	MPDZ66	Multiple PDZ domain protein (MUPP1)	Associated with tight junctions;
E7		F-actin67	Cytoskeletal component
Apoptosis
E6	TP5368	Tumor protein p533	“Guardian of the genome”; apoptosis; cell cycle control
	TP7369	Tumor protein p73	Cell cycle regulation; apoptosis
	BAK170	BCL2-antagonist/killer 1	Promotes apoptosis
	FADD71	Fas (TNFRSF6)-associated via death domain	Apoptosis
	TNFRSF1A72	Tumor necrosis factor receptor superfamily, member 1A	Apoptosis; inflammation
E7	PPP2CA73	Protein phosphatase 2 (formerly 2A), catalytic subunit, alpha isoform	Dephosphorylates protein kinase B/Akt; cell survival;
	SIVA74	SIVA 1, apoptosis-inducing factor	Promotes apoptosis
	PML75	Promyelocytic leukemia	Cellular senescence
	DNAJA376	Dna J (Hsp 40) homolog, subfamily A, member 3	Co-chaperone protein; regulates NF(B activity
	IGFBP377	Insulin-like growth factor binding protein-3	Apoptosis; cell cycle regulation
Cell Cycle
E6	TSC278	Tuberous sclerosis 2	S6 kinase inhibitor; inhibits cell growth; Akt phosphorylates tuberin inhibiting cellular senescence
	MCM779	Minichromosome maintenance complex component 7	DNA replication
E7	CDKN1A80	Cyclin-dependent kinase inhibitor 1A (p21, Cip1)	Cyclin-dependent kinase inhibitor
	CDKN1B81	Cyclin-dependent kinase inhibitor 1B (p27, Kip1)	Cyclin-dependent kinase inhibitor
	CDK282	Cyclin-dependent kinase 2	Cell cycle regulation
	CDKN2A†83	Cyclin-dependent kinase inhibitor 2A (melanoma, p16, inhibits CDK4)	Cyclin-dependent kinase inhibitor
	E2F1/CCNA284	E2F transcription factor/cyclin A2	Cell cycle regulation
		Histone H1 kinase85	Cell cycle regulation
	CCNA182	Cyclin A1	Cell cycle regulation
	CCNE186	Cyclin E1	Cell cycle regulation; transcriptional regulation
	RB187	Retinoblastoma 1 (including osteosarcoma)	Regulates E2F transcription factor; cell cycle regulation
DNA Repair
E6	XRCC136	X-ray repair complementing defective repair in Chinese hamster cells 1	Single-strand break repair
	MGMT88	O-6-methylguanine-DNA methyltransferase	Repairs alkylation damage
E6/E7	BRCA137	Breast cancer 1, early onset	DNA repair; transcriptional regulator
Metabolism
E7	PSMC189	Proteasome (prosome, macropain) 26S subunit, ATPase, 1	Protein turnover
	PKM290	Pyruvate kinase, muscle	Glycolytic enzyme
	NME191	Nonmetastatic cells 1, protein (NM23A) expressed in	Metastasis suppressor; nucleoside diphosphate kinase;
	NME291	Nonmetastatic cells 2, protein (NM23B) expressed in	Metastasis suppressor; nucleoside diphosphate kinase
Signal Transduction
E6	GIPC192	GIPC PDZ domain containing family, member 1	Transforming growth factor-β signaling; G-protein coupled receptor signaling
	TAX1BP393	Tax 1 (human T-cell leukemia virus type 1) binding protein 3	Rho signaling; inhibits β-catenin function; scaffold-interacting protein
	SIPA1L194	Signal-induced proliferation-associated 1 like 1	Rap GTPase-activating protein (GAP)
	TYK295	Tyrosine kinase 2	Interferon signaling; tyrosine kinase
	PTPN396	Protein tyrosine phosphatase, non-receptor type 3	Membrane-associated protein tyrosine phosphatase
	PKN197	Protein kinase N1	MAP kinase signaling
E7	CHUK IKBKB98	Conserved helix-loop-helix ubiquitous kinase/inhibitor of kappa light polypeptide gene enhancer in B-cells, kinase beta	Regulates IκB phosphorylation
Transcription
E6	IRF399	Interferon regulatory factor-3	Transcriptional activator of interferon and interferon-inducible gene promoters
	NFX1100	Nuclear transcription factor, X-box binding 1	Transcriptionally represses hTERT promoter
	TADA3L101	Rranscriptional adaptor 3 (NGG1 homolog, yeast)-like	Stabilizes TP53; transciptional coactivator with TP53 and histone acetyltransferases
	SERTAD1102	SERTA domain containing 1	Transcriptional regulator; cell cycle
E6/E7	TAF9103	TAF9 RNA polymerase II, (TBP)-associated factor, 32 kDa	Transcriptional coactivator
	SMAD3104, 105	SMAD family member 3	Transforming growth factor-β signaling; transcription factor
	CREBBP106, 107	CREB binding protein (Rubinstein-Taybi syndrome)	Transcriptional coactivator; binds to pCAF; histone acetyltransferase
	MYC108, 109	v-myc myelocytomatosis viral oncogene homolog (avian)	Apoptosis; transcription factor
E7	RBL1110	Retinoblastoma-like 1 (p107)	Transcriptional regulation
	RBL2110	Retinoblastoma-like 2 (p130)	Transcriptional regulation
	TBP111	TATA box binding protein	Transcriptional regulation
	JUN112	jun oncogene	Transcription factor
	FHL2113	4.5 LIM domains 2	Transcriptional coactivator
	DNMT115	DNA (cytosine-5-)-methyltransferase 1	DNA methyltransferase
	HDAC1114	Histone deacetylase 1	Histone deacetylase; regulates transcription
	SNW1115	SNW domain containing	Transcriptional coactivator
	IRF1116	Interferon regulatory factor 1	Transcriptional activator of interferon and interferon-inducible gene promoters
	MPP2117	Membrane protein, palmitoylated 2 (MAGUK p55 subfamily member 2)	Transcription factor
	PCAF14	P300/CBP-associated factor	Histone acetyltransferase
	ISGF3G118	Interferon regulatory factor 9	DNA-binding component of interferon-stimulated gene factor 3 (ISGF3)
Unknown/Other Function
E6	GOPC119	Golgi associated PDZ and coiled-coil motif containing	Regulates trafficking of cystic fibrosis transmembrane conductance regulator (CFTR)
	RCN2120	Reticulocalbin 2, EF-hand calcium binding domain	Calcium-binding protein
	FBLN1121	Fibulin 1	Calcium-binding extracellular matrix protein
	E6AP122, 123	E6-associated protein	E3 Ubiquitin ligase
E7	TAP1124	Transporter 1, ATP-binding cassette, sub-family B (MDR/TAP1)	Major Histocompatibility Complex (MHC) class 1 antigen presentation
	TUBG1125	Gamma-tubulin	Centrosome-associated protein

Complex cytogenetic and epigenetic changes accumulate with neoplastic progression. In cervical cancer, gain of chromosome 3q is one of the earliest and most consistent findings.8 Epigenetic alterations regulate gene expression without changing the DNA sequence. These alterations include methylation of cytosine in DNA and acetylation of histone proteins. Epigenetic alterations are increasingly recognized as important oncogenic mechanisms, and HPV-associated oncogenesis is no exception (Table 2). DNA methylation is also presumed to be a cellular defense to silence foreign DNA transcription.9 Methylation of HPV is known to occur. Recent studies of HPV type 16 (HPV-16) and HPV-18 have shown that the extent of methylation varies by region of the genome, and changes in the methylation patterns may be associated with grade of neoplasia.10–13 Epigenetic changes could also occur through direct interaction between HPV and cellular proteins involved in DNA methylation and chromatin remodeling, including histone deacetylase (HDAC), DNA methyltransferase (DNMT), and p300 (Table 1).14, 15

Table 2. Epigenetic Alterations in HPV-Associated Cervical Cancer*

Cellular Process§		Methylation of Invasive Cervical CarcinomaVersus Normal Tissue†						Methylation in Premalignant Lesions‡
		Normal			ICC			LSIL			HSIL
		No.	%	(Range)	No.	%	(Range)	No.	%	(Range)	No.	%	(Range)
HPV indicates human papillomavirus; ICC, invasive cervical cancer; LSIL, low-grade squamous intraepithelial lesion; HSIL, high-grade intraepithelial lesion; kD, kilodalton; E. coli, Escherichia coli. * Data presented in Table 2 are a summary of methylation profiling studies in the literature. Data are based on heterogeneous methylation assessment methods and heterogeneous sample sets (tissue and cervical cells), and the functional verification of published findings has not been conducted for many of the genes examined in clinical samples. † Median % (% range) of samples reported methylated within normal cervical tissue and invasive cervical cancer (squamous cell carcinoma and adenocarcinoma combined) across studies. ‡ Median % (% range) of samples reported as methylated within LSIL (cervical intraepithelial neoplasia [CIN] type 1) and HSIL (CIN2/3). Published data are not available for many genes. § The majority of genes contribute to multiple cellular processes.
Adhesion
CDH157, 127, 129, 130, 134, 136, 138, 148, 150, 151, 158, 171	Cadherin 1, type 1, E-cadherin (epithelial)	11	0	(0-100)	11	53	(0-91)	2	0	(0-0)	3	17	(0-39)
THBS1136, 138, 150	Thrombospondin 1	1	0		3	38	(35-46)
CDH13130, 158	Cadherin 13, H-cadherin (heart)	2	14	(8-20)	2	64	(46-82)	1	4		2	14	(13-16)
CADM1145, 155, 171	Cell adhesion molecule 1	3	0	(0-0)	2	62	(58-65)	2	0	(0-0)	2	49	(35-63)
DLC1152	Deleted in liver cancer 1				1	87
SLIT2147	Slit homologue 2 (Drosophila)	1	0		1	64		1	2		1	25
SLIT1147	Slit homologue 1 (Drosophila)	1	0		1	53		1	0		1	10
SLIT3147	Slit homologue 3 (Drosophila)	1	0		1	49		1	4		1	2
ROBO1147	Roundabout, axon guidance receptor, homologue 1 (Drosophila)	1	0		1	46		1	7		1	8
ROBO3147	Roundabout, axon guidance receptor, homologue 3;	1	0		1	36		1	0		1	10
CTNNB157	Catenin (cadherin-associated protein), beta 1, 88 kD	1	0		1	0
Apoptosis
DAPK157, 129, 130, 134, 136, 138, 148, 150, 158, 162, 163, 170, 171	Death-associated protein kinase 1	10	0	(0-8)	12	58	(45-82)	2	4	(0-8)	3	35	(23-64)
TIMP357, 134, 136, 138, 148, 150, 158	TIMP metallopeptidase inhibitor 3 (Sorsby fundus dystrophy, pseudoinflammatory)	5	0	(0-0)	7	10	(1-100)				1	16
TNFRSF10C154	Tumor necrosis factor receptor superfamily member 10c, decoy without an intracellular domain				1	100
PYCARD130	PYD and CARD domain containing	1	3		1	7		1	3		1	0
TNFRSF10D154	Tumor necrosis factor receptor superfamily member 10d, decoy with truncated death domain				1	18
Cell Cycle
CDKN2A129, 130, 133, 134, 135, 136, 138, 139, 144, 146, 148, 149, 150, 157, 160, 162, 163, 167, 168, 171	Cyclin-dependent kinase inhibitor 2A (melanoma, p16, inhibits CDK4)	12	2	(0-21)	18	27	(3-61)	8	28	(0-50)	8	30	(0-55)
APC57, 129, 130, 136, 138, 148, 150, 170	Adenomatosis polyposis coli	10	1	(0-41)	12	15	(1-46)	3	0	(0-4)	4	5	(0-18)
FHIT57, 130, 136, 148, 151, 157, 161	Fragile histidine triad gene	6	0	(0-39)	7	20	(11-54)	1	32		1	34
RASSF157, 130, 136, 137, 138, 142, 143, 148, 150, 158, 164, 169, 171	Ras association (RalGDS/AF6) domain family 1	7	0	(0-94)	7	32	(0-79)	2	1	(0-3)	2	1	(0-2)
HIC157, 129, 130, 148, 171	Hypermethylated in cancer 1	5	0	(0-64)	4	32	(4-71)	2	46	(24-68)	2	48	(30-67)
TERT22, 158, 159	Telomerase reverse transcriptase;	3	0	(0-27)	2	70	(59-82)	1	68		2	39	(0-78)
PTEN128, 162, 171	Phosphatase and tensin homologue (mutated in multiple advanced cancers 1)	1	0	(0-0)	2	37	(16-58)	1	0		2	20	(0-40)
SFN130	Stratifin	1	94		1	99		1	97		1	97
CCNA1141	Cyclin A1	1	0		1	93		1	0		1	36
HSPA2158	Heat-shock 70-kD protein 2	1	0		1	73					1	3
ESR157	Estrogen receptor 1	1	0		1	32
(continued)
Adhesion
RPRM156	Reprimo, TP53 dependent G2 arrest mediator candidate				1	18
CCND2130, 171	Cyclin D2	2	2	(0-4)	1	7		2	0	(0-0)	2	0	(0-0)
CDKN2B130, 171	Cyclin-dependent kinase inhibitor 2B; p15	2	1	(0-2)	1	4		2	1	(0-3)	2	1	(0-2)
VHL130	von Hippel-Lindau tumor suppressor	1	1		1	2		1	0		1	2
DNA Repair
MGMT129, 130, 136, 147, 148, 150, 157, 162, 163, 170, 171	O-6-methylguanine-DNA methyltransferase	9	0	(0-15)	10	18	(5-40)	4	7	(0-30)	4	15	(0-35)
MLH157, 130, 136, 138, 148, 150, 157, 158, 171	mutL homolog 1, colon cancer, nonpolyposis type 2 (E. coli)	7	0	(0-9)	8	2	(0-36)	3	6	(0-14)	4	5	(2-18)
BRCA1148	Breast cancer 1, early onset	1	0		1	6
TP73148, 171	Tumor protein p73	2	9	(0-18)	1	0		1	12		1	18
Signal Transduction
RARB57, 130, 131, 148, 157, 165, 171	Retinoic acid receptor, beta	7	0	(0-15)	6	34	(18-69)	3	0	(0-0)	3	9	(9-29)
HLTF136, 138	Helicase-like transcription factor	1	0		2	10	(4-16)
SOCS2158	Suppressor of cytokine signaling 2	1	23		1	64					1	45
SOCS1158	Suppressor of cytokine signaling 1	1	0		1	50					1	7
TIMP2132	TIMP metallopeptidase inhibitor 2	1	11		1	47
TYRO3130	TYRO3 protein tyrosine kinase	1	6		1	15		1	0		1	4
CAV1126	Caveolin 1, caveolae protein, 22 kD	1	0		1	6
Transcription
RUNX3136, 140, 150	Runt-related transcription factor 3	1	0		3	2	(0-3)
TWIST1130	Twist homologue 1 (acrocephalosyndactyly 3; Saethre-Chotzen syndrome) (Drosophila)	1	0		1	43		1	0		1	14
POU2F3166	POU domain, class 2, transcription factor 3	1	0	(0-0)	1	39
PRDM2130	PR domain containing 2, with ZNF domain	1	2		1	3		1	0		1	0
Other/Unknown Processes
GSTP1130, 136, 148, 150, 157, 158, 170, 171	Glutathione S-transferase pi	7	0	(0-13)	7	0	(0-21)	3	0	(0-0)	4	5	(0-18)
PTGS2136, 150	Prostaglandin-endoperoxide synthase 2 (prostaglandin G/H synthase and cyclooxygenase)	1	0		2	14	(13-15)
ZMYND10143	Zinc finger, MYND-type containing 10	1	13		1	71		1	20		1	57
CALCA57	Calcitonin/calcitonin-related polypeptide, alpha	1	0		1	68
SCGB3A1153	Secretoglobin, family 3A, member 1				1	18

Cellular Processes Affected by HPV

Apoptosis and cell cycle control

The most recognized target of HPV E6 protein is TP53,16 whereas the primary target of HPV E7 protein is RB1 and the related pocket proteins, p107 and p130.17 E6 binding to TP53 leads to inactivation and degradation of TP53. Degradation of TP53 and other proteins bound to HPV E6 occurs through the ubiquitin pathway, mediated by a cellular protein named E6-associated protein (E6AP). E6AP is an E3 ubiquitin ligase; the E3 nomenclature refers to the ubiquitin pathway and has nothing to do with HPV early genes. E7 binding to RB1 results in inactivation of RB1 functions, including release of a transcription factor, E2F, and destabilization of RB1. The overall effect of E6/E7 targeted activity is the loss of apoptosis and genome guarding by TP53 and loss of cell cycle control by RB1. Numerous other tumor suppressor genes involved in cell cycle control and apoptosis are also targeted by E6/E7 and methylation (Tables 1 and 2).

Changes in TP53 and hTERT, the reverse transcriptase catalytic component of telomerase, activity and the consequent loss of apoptosis are frequently associated with immortalization and cancer progression. McMurray and McCance18 suggested that degradation of TP53 may be the principal mechanism responsible for immortalization in HPV E6/E7-expressing cells and that hTERT activation may be dispensable. C-myc and NFX1-91 (Table 1) are 2 transcription factors directly targeted by HPV oncoproteins, the former resulting in transcriptional activation and the latter resulting in transcriptional repression of the hTERT gene.19, 20 The RNA, noncatalytic component of telomerase, hTERC, has not been demonstrated to interact directly with HPV oncoproteins; however, increased copy number has been reported with a gain of chromosome 3q.21 Epigenetic regulation of hTERT through histone modification has been reported after the interaction between E6, E6AP, and p300, a coactivator/acetyl transferase.21 Interestingly, telomerase is hypermethylated during progression to invasive cervical cancer (Table 2); however, hTERT expression and activity did not correlate with methylation.22

DAP kinases (DAPK) are a family of proapoptotic, Ca²⁺/calmodulin-regulated, serine/threonine kinases that also control autophagic cell death.23, 24 DAPK has been consistently methylated in squamous cell carcinoma (SCC) (47%-77%) and in high-grade squamous intraepithelial lesions (HSILs) (23%-64%), but it is usually unmethylated in normal cells (0%-8%) and low-grade squamous intraepithelial lesions (LSILs) (0%-8%). The loss of DAP kinase expression in premalignant lesions (eg, HSIL) may influence the progression to invasive cervical cancer (Table 2).

Cell adhesion

Cancer progression is characterized by a disruption of cell cohesion and consequent breakdown of normal communication between neighboring cells. The human homologs of the Drosophila tumor suppressors Dlg and Scribble (hDlg and hScrib) are cell adhesion proteins that interact with adenomatous polyposis coli (APC) and are targeted for degradation by HPV E6.25, 26 Inhibiting the APC/hScrib interaction disrupts adherens junctions important in cell adhesion,25 whereas loss of the hDlg/APC interaction reduces the negative regulation of entry into S-phase of the cell cycle.26 In common with many other cell adhesion proteins, hDlg and hScrib contain PDZ domains. The PDZ domain was first identified in postsynaptic density protein (PSD-95), Discs-large protein (Dlg), and the epithelial tight-junction protein, ZO-1.27 The E6 protein of only oncogenic, and not nononcogenic, HPV contains a PDZ-binding motif located at the extreme end of the carboxy terminus.28

In addition to several other adhesion proteins, E-cadherin (CDH1) is frequently methylated in cervical cancer. CDH1, a component of cellular adherens junctions and tight junctions, sequesters β-catenin at the cell membrane.29 Loss of CDH1 releases β-catenin into the cytoplasm and disrupts signaling by the WNT pathway.30 The loss of E-cadherin expression has been shown to result in reduced numbers of Langerhans cells within CIN and invasive cervical cancer31 and increased invasiveness in vitro.32 Recent studies suggest an effect of HPV on WNT signaling.33, 34

DNA repair

DNA repair is an important cellular mechanism for maintaining genomic stability, and the development of genomic instability is universal among neoplastic cells. DNA methylation changes have also been associated with the development of genomic instability.35 HPV E6/E7 proteins induce genomic instability indirectly through loss of cell cycle control when DNA repair is needed, and directly through binding to proteins involved in DNA repair. HPV E6 binds to XRCC1, a scaffolding protein involved in repairing single-strand DNA breaks, interfering with DNA repair activities.36 HPV oncoproteins have been reported to interact with BRCA1 and antagonize its function, but they do not degrade BRCA1.37 BRCA1 has many cellular functions in addition to DNA repair,38 including transcriptional regulation of hTERT39 and TP5340 and RB1-mediated cell cycle control.41

Molecular Biomarkers: Translation to Public Health

For the uterine cervix, the Papanicolaou test (Pap test) has been used for decades to identify malignant and premalignant lesions through cytologic examination. The Pap test demonstrates that early detection and treatment of preinvasive disease prevents invasive cancer. Countries that widely employ Pap screening have experienced dramatic declines in cervical cancer incidence and mortality.

Because current formulations of the HPV vaccine do not protect against all neoplastic types, screening will remain an important part of cervical cancer prevention. The Pap test approach is unlikely to address all future screening needs adequately. Limitations in the sensitivity of a single Pap test require that screening be done at regular intervals and that women with abnormal screening be recalled for definitive diagnosis and treatment. Currently, fewer than one-third of women referred for colposcopy have confirmed disease that requires treatment. In addition, the introduction of HPV vaccines can be anticipated to further reduce cytologic abnormalities associated with true cervical neoplasia, whereas having a lesser impact on nonspecific or mild screening abnormalities. Unless the sensitivity of screening is improved, the cost and inefficiency of screening will increase and erode the cost-effectiveness of vaccination.

Appreciation of the central role of HPV in oncogenesis led to the introduction of HPV testing as a triage for women with equivocal cytology results42 and then as an adjunct or supplement to Pap test screening.42–45 Therefore, HPV represents one of the first examples of a screening biomarker. Although HPV testing provides a sensitive screening tool, a significant number of women without cervical disease test positive for oncogenic HPV.46 To achieve the goal of efficient screening, limitations in the specificity (ie, the high percentage of false-positive tests) of HPV testing need to be addressed. It needs to be emphasized that there are currently no clinical recommendations for primary screening by use of HPV testing in the US.

The increased understanding of molecular changes associated with cervical neoplasia, as indicated in this review, has prompted investigators to examine additional molecular biomarkers as surrogates for neoplastic progression. These efforts have been summarized in recent reviews.47–49 Some of these approaches are highlighted below.

CDKN2A (p16^INK4A) is increased in HPV oncogenesis by the combined effect of E6 and E7 (Table 1), and thus it is viewed as a surrogate for oncogenic HPV. Immunohistochemical detection of CDKN2A has shown promise in the evaluation of equivocal cervical Pap testing in diverse populations from around the world,50–53 although large-scale studies are lacking. CDKN2A staining correlates with neoplastic progression in cervical biopsies; CIN 1 lesions typically stain more weakly than high-grade lesions, and CDKN2A is usually undetectable in normal tissues. Direct comparison of CDKN2A immunoreactivity and oncogenic HPV detection as predictors of high-grade cervical lesions and cancer suggests that CDKN2A improves specificity at the cost of decreased sensitivity, although some studies suggest that the sensitivity of the CDKN2A test can approach 80% to 90%.51, 52

Immunohistochemical biomarkers may be used to improve the efficacy of the Pap test, an approach that may demonstrate the most impact in areas of the world that have both the greatest burden of cervical disease and the fewest resources and trained personnel to implement routine standard Pap testing. For example, Mukherjee et al54 tested the performance of initial screening of Pap test smears with immunohistochemistry for minichromosome maintenance protein (MCM)-2 and -5 versus standard cytology screening in Bangalore, India. The MCM test required less skill to interpret than the standard Pap test. They found that the MCM test was significantly faster and had greater interobserver agreement than the Pap test. All patients in this study who displayed abnormalities on Pap testing were found to be MCM positive. These results suggest that initial screening of underserved populations with MCM may be as effective as Pap testing, but significantly easier to implement. Larger population-based studies are needed. Interestingly, MCM was one of the first molecular markers used on Pap test material as a ‘proof of concept’ for molecular screening,55 and it has been used in combination with TOP2A, Topoisomerase (DNA) IIα, in a newly marketed test.48

To our knowledge, few studies have been performed to date to evaluate the efficacy of using changes in DNA methylation for the diagnosis of high-grade cervical lesions and cancer. A current research focus is on understanding the biologic significance of L1 methylation of the viral genome, reported as a sensitive biomarker for cervical dysplasia.11 L1 methylation might be expected, given that the viral life cycle is progressively abbreviated during cancer progression so that in cervical cancer L1 expression is usually minimal or completely lost.56 Confirmatory studies are needed. In terms of host cellular gene methylation, it appears that the greatest sensitivity and specificity may be attained by using a panel of methylated genes. In one study, a panel of 4 methylated genes consisting of CALCA, DAPK, ESR1, and APC yielded a sensitivity and specificity of 89% and 100%, respectively, for the determination of cervical adenocarcinoma.57 Further large-scale studies are needed to fully determine the widespread applicability of this approach.

To our knowledge, other than oncogenic HPV, molecular tests for cervical cancer screening have not been widely validated. Several problems may contribute to the limited success to date. In screening samples <1% of the cells may be abnormal, so that approaches that use bulk DNA or RNA extracts have the technical challenge of signal dilution from the normal cells in the sample. The histologic endpoint of CIN 3 may not represent molecularly homogenous endpoints. The biologic potential of these lesions may vary, and there could be multiple different pathways that lead to the same histology. Although studies in prospective cohorts are difficult and expensive, additional translational research in such cohorts is required for marker validation.