looking in the porphyromonas gingivalis cabinet of curiosities the microbium, the host and cancer...

INVITED REVIEW

Looking in the Porphyromonas gingivalis cabinetof curiosities: the microbium, the host andcancer associationK.R. Atanasova1 and O. Yilmaz1,2

1 Department of Periodontology, University of Florida, Gainesville, FL, USA2 Emerging Pathogens Institute, University of Florida, Gainesville, FL, USA

Correspondence: Ozlem Yilmaz, Department of Periodontology, College of Dentistry and Emerging Pathogens Institute, University of Florida,Gainesville, FL 32610-0434, USA Tel.: + 1 352 273 8003; fax: + 1 352 273 9399; E-mail: [email protected]

Keywords: cancer; cellular; molecular; oral microbiology; PorphyromonasAccepted 16 January 2014DOI: 10.1111/omi.12047

SUMMARY

The past decades of biomedical research haveyielded massive evidence for the contribution ofthe microbiome in the development of a variety ofchronic human diseases. There is emerging evi-dence that Porphyromonas gingivalis, a well-adapted opportunistic pathogen of the oralmucosa and prominent constituent of oral bio-lms, best known for its involvement in periodon-titis, may be an important mediator in thedevelopment of a number of multifactorial andseemingly unrelated chronic diseases, such asrheumatoid arthritis and orodigestive cancers.Orodigestive cancers represent a large proportionof the total malignancies worldwide, and includecancers of the oral cavity, gastrointestinal tractand pancreas. For prevention and/or enhancedprognosis of these diseases, a good understand-ing of the pathophysiological mechanisms andthe interaction between P. gingivalis and host ismuch needed. With this review, we introduce thecurrently accumulated knowledge on P. gingiva-liss plausible association with cancer as a riskmodier, and present the putative cancer-promot-ing cellular and molecular mechanisms that thisorganism may inuence in the oral mucosa.

Knowledge is made by oblivion, and to purchase aclear and warrantable body of truth, we must forgetand part with much we know.

Sir Thomas Browne (16051682)

INTRODUCTION

The last few decades of biomedical research haveaccumulated a large knowledge base on the etiopath-ogenic mechanisms of numerous chronic diseasesand conditions of man. In many of these chronicpathologies, bacteria have been shown to play amajor role either as singular etiological factors, or assynergistic contributors in the multifaceted stimulationof the host immune system that can tip the scalestowards chronic or autoimmune disease development.Recent advances in metagenomic tools have height-ened the importance of the resident microbiome inmammalian development, metabolism, immuneresponse and behavior during all stages of life, whichhas led to the initiation of several world-wide projectsto determine the species diversity and genetic make-up of the human microbiome in health and disease(Diaz Heijtz et al., 2011; Cox et al., 2013; Erturk-Hasdemir & Kasper, 2013; Guinane & Cotter, 2013;

molecular oral microbiology

2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd 55Molecular Oral Microbiology 29 (2014) 5566

molecular oral microbiology

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Mavrommatis et al., 2013; Ridaura et al., 2013). Mul-tiple specic examples may be given for the growingrole of the human microbiome in the development ofchronic pathological conditions. These include chronicinammatory diseases of the lung, liver, cardiovascu-lar and digestive systems, as well as of periodontium(Berry & Reinisch, 2013; Chassaing et al., 2013). Thehuman microbiome contains a number of adaptablepathogenic species that are now identied as beingclosely involved in the etiology of various chronic dis-eases. One of the best documented examples is agram-negative bacterium, Helicobacter pylori, primar-ily known for its involvement in gastric and gastroin-testinal cancers, and more recently in other chronicdiseases, including asthma, type 2 diabetes andobesity (Wang et al., 2013). Furthermore, increasingevidence suggests that the establishment of a varietyof chronic diseases could result from polymicrobialinteractions within the underlying microbiome, in con-cert with host genetic and metabolic risk factors(Wroblewski et al., 2010; Fox & Sheh, 2013;Michaud, 2013).The human body represents a multitude of mucosal

sites, each comprising a specic milieu of highlyadapted, commensal and opportunistic-pathogenorganisms. One of the most complex and highly vari-able communities is the oral microbiome, which haslong been implicated in the development of toothdecay and periodontal disease (Jenkinson, 2011).The oral microbiome represents a unique and vastlydynamic multidimensional biochemical network,formed by the interaction among the proteomes andmetabolomes of the comprising organisms, thebehavioral, hygienic and dietary habits of the host,and the genetic and immunological host factors (Kha-lili, 2008; Jenkinson, 2011; Teles et al., 2013; Wrightet al., 2013). Some of these interactions yield poten-tially pathogenic compounds that may have effects onthe mucosal lining, the surrounding tissues and evenon the distant organs and systems of the body (Hoo-per et al., 2009; Jenkinson, 2011; Ahn et al., 2012a).Those noxious metabolic products or moleculesinclude lipids, lipopolysaccharides, free oxygen andnitrogen radicals, pro-inammatory mediators andhost and/or bacteria-derived enzymes. Hence, it hasbeen proposed that the oral microbiome impacts onthe etiology and progression of diabetes and cardio-vascular disease, and recently a number of studieshave indicated the association among distinct

consortia of microbial species, periodontal diseaseand orodigestive cancers (Meyer et al., 2008; Korenet al., 2011; Ahn et al., 2012a). The evidence accu-mulated from those studies has specied strong cor-relation among a number of chronic periodontalbacteria containing Prevotella, Porphyromonas, Fuso-bacterium and Streptococcus spp., and squamouscell carcinoma of the orodigestive tract, which encom-passes the oral cavity, gastrointestinal tract and pan-creas (Nagy et al., 1998; Mager et al., 2005; Ahnet al., 2012b).Among these specic species, an opportunistic

pathogen, Porphyromonas gingivalis, has emerged asa potential mediator in the etiology of a variety of pre-sumably unrelated chronic diseases, such as rheuma-toid arthritis, cardiovascular diseases, diabetes and,more recently, different types of orodigestive cancers(Hitchon et al., 2010; Katz et al., 2011; Ahn et al.,2012b; Hajishengallis et al., 2012; Aemaimananet al., 2013; Ishikawa et al., 2013; Michaud, 2013;Totaro et al., 2013). In the case of rheumatoid arthri-tis, the P. gingivalis enzyme, peptidylarginine deimin-ase, has been recently suggested to assume a keyrole in the citrullination of arginine residues in hostproteins, so altering their normal folding and creatingconditions for the induction of auto-antibodies againsthost cartilage-specic antigens. The ability of P. gingi-valis to activate host matrix metalloproteases (MMPs)and the organisms role in altering cytokine responseshave also been implicated in the protein degradation,observed in arthritic joints (Detert et al., 2010; Hitchonet al., 2010; Mangat et al., 2010). Conjointly, pro-longed systemic cytokine elevation and inammationhave been especially attributed to P. gingivalis in thecontext of the multifactorial periodontal disease ascontributing and/or amplifying factor in the develop-ment and progression of diabetes mellitus. The poorglycemic control in diabetes, on the other hand, hasbeen shown to exacerbate chronic periodontitis (Lalla& Papapanou, 2011). A signicant statistical associa-tion has also been observed between P. gingivalis-carriage in saliva samples and non-alcoholic fattyliver disease, further strengthening P. gingivalis roleas a risk factor in multiple chronic conditions (Yonedaet al., 2012).On the cancer front, multiple clinical and experi-

mental studies have displayed various degrees ofassociations between P. gingivalis and cancers of theoral cavity, orodigestive tract or pancreatic tissues

56 2014 John Wiley & Sons A/S. Published by John Wiley & Sons LtdMolecular Oral Microbiology 29 (2014) 5566

P. gingivalis and cancer K.R. Atanasova and O. Yilmaz

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(Groeger et al., 2011; Katz et al., 2011; Ahn et al.,2012b; Michaud et al., 2012; Michaud, 2013).Porphyromonas gingivalis, which has been shown

to have an abundance of virulence factors, can alsobe present in healthy individuals periodontal pockets.It afuently survives within oral epithelial tissues, andis successfully able to evade the host immuneresponse (Yilmaz, 2008; Yilmaz et al., 2008, 2010;Yao et al., 2010; Singh et al., 2011; Choi et al.,2013). Accordingly, P. gingivalis has recently beenattributed as a key-stone pathogen based on its abil-ity to orchestrate inammatory disease by remodelingthe oral microbiome (Hajishengallis et al., 2012). TheP. gingivalis can invade primary cultures of oral epi-thelial cells (OECs), render them resistant to celldeath, and alter the chemokine and cytokine secre-tion (Yao et al., 2010; Yilmaz et al., 2010; Spooner &Yilmaz, 2011; Choi et al., 2013; Hung et al., 2013;Takeuchi et al., 2013). OECs that form the outer lin-ing of the oral mucosa are the initial targets for colo-nizing bacteria, and form an important arm of the hostinnate immune response by secreting antimicrobial,pro-inammatory and chemotactic signals, and modu-lating apoptosis upon sensing invading organisms(Yilmaz et al., 2010; Hung et al., 2013; Takeuchi

et al., 2013). Therefore, the ability of P. gingivalis tocolonize in the oral cavity and modulate both the epi-thelial cell and systemic responses potentially placesthe organism not only in the category of central con-tributors in the multifactorial etiology of the periodon-tal disease, but also orodigestive cancers and otherchronic diseases (Fig. 1).The prospective multiple roles of P. gingivalis in the

above-mentioned conditions, however, remain incom-plete and there is a clear need for mechanistic deter-mination of these newly observed associations tobetter understand and possibly control those severechronic conditions. Therefore, this review aims tooffer a fresh perspective on the putative role ofP. gingivalis in the orodigestive cancers, and its plau-sible use as a biomarker for the identication of athigh-risk populations.

Porphyromonas gingivalis and cancer

The signicance of P. gingivalis involvement in can-cer has evolved within the last decade. Chronologi-cally initial studies have comprised mostlyepidemiological and clinical association between oralmicrobiome, periodontal disease or tooth loss, with

Figure 1 Schematic representation of the complex interrelationships between different human genetic, behavioral and immunological factors,as well as microbiome-related factors, that are proposed to take part in the multifactorial etiology of orodigestive cancers and other possiblyassociated chronic diseases. Porphyromonas gingivalis is implicated to play a specic role in these multi-directional links. Dashed arrows rep-

resent plausible associations, based on currently available epidemiological, clinical, histological and experimental studies. Abbreviations: NF-jB, nuclear factor-jB; ROS, reactive oxygen species.


K.R. Atanasova and O. Yilmaz P. gingivalis and cancer

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orodigestive cancers including cancers of the oralcavity, gastrointestinal tract and pancreas (Meyeret al., 2008; Hooper et al., 2009; Groeger et al.,2011; Katz et al., 2011; Ahn et al., 2012a,b; Michaud,2013). Among all observed cancer relations the mostdirect and strong association of P. gingivalis hasbeen found with oral squamous cell carcinoma(OSCC) (Nagy et al., 1998; Mager et al., 2005; Katzet al., 2011). OSCC is a common cancer in the USAwith an estimated 35,000 newly diagnosed cases andover 7500 deaths occurring yearly (Jemal et al.,2008). Currently there is a lack of reliable diagnostictools for identication of high-risk persons, mostOSCC cases have metastases already at presenta-tion, and available therapies fail to prevent malignantprogression (Mitka, 2013). All these factors supportthe necessity for early markers that can identify therisk populations and possibly be used for preventivecare, before carcinogenesis develops.

HISTORICAL PERSPECTIVE AND CLINICALRELEVANCE

Among the rst studies to examine associationbetween periodontitis and cancer was a casecontrolstudy conducted between 1999 and 2005 in the USAthat examined 473 patients (Tezal et al., 2009). Thestudy measured periodontitis by alveolar bone lossand screened the patients for squamous cell carci-noma of the oral cavity, pharynx and larynx. Chronicperiodontitis was associated with four-fold increase inrisk of any of the three examined types of carcinoma.The strength of the association was greatest withcancers of the oral cavity, followed by the oropharynxand larynx. The association persisted in subjects whonever used tobacco and alcohol, which suggests thatchronic periodontitis is an independent risk factor forthe development of cancer (Tezal et al., 2009).One of the initial broad surveillance studies show-

ing a potential link between P. gingivalis and orodi-gestive cancers was the National Health and NutritionExamination Survey III (Ahn et al., 2012b). This pro-spective study included a total of 12,605 men andwomen with clinically ascertained periodontitis ofwhom 7852 had a serum IgG immune response toP. gingivalis. They were followed from 1988 through2006 in relation to orodigestive cancer mortality,including cancers of the lip, oral cavity, gastrointesti-nal tract, pancreas and liver, and in that period 105

orodigestive cancer deaths occurred. Moderate orsevere periodontitis was found to be associated withincreased orodigestive cancer mortality relative risk.The mortality risk was also proportionally increasedwith increasing severity of periodontal disease. Thehighest association with periodontitis was found forcolorectal and pancreatic cancer, whereas greaterserum P. gingivalis IgG levels tended to be associ-ated with increased orodigestive cancer mortality(Ahn et al., 2012b). Interestingly, P. gingivalis wasalso associated with 2.25-fold higher likelihood of oro-digestive mortality in healthy subjects not exhibitingovert periodontal disease. Hence, this study was therst to illustrate direct correlation of orodigestive can-cer mortality to P. gingivalis independently of peri-odontal disease, so pinpointing that P. gingivaliscould be a valuable biomarker for microbe-associatedrisk of orodigestive cancer death (Ahn et al., 2012b).In 1998, a study compared biolm samples from

central ulcerating surfaces of OSCC lesions andhealthy mucosa from 21 middle-aged, mostly malepatients without antibiotic or tumor therapy (Nagyet al., 1998). The study found a signicantly highernumber of both specic anaerobic and aerobic bacte-rial species at the tumor sites compared with healthymucosa. Interestingly the genus Porphyromonas wasidentied among the genera with highest rates of iso-lation at the tumor sites (Nagy et al., 1998). Morethan a decade later, in 2012, a similar study quanti-ed the 16S ribosomal DNA fragments from sixperiodontal bacteria including P. gingivalis in formalin-xed, parafn-embedded lymph nodes from 66patients with histories of head and neck cancers,including OSCC (Amodini Rajakaruna et al., 2012).The lymph nodes selected for the study did not havemetastatic invasion yet. The relationship betweenbacterial detection and cancer severity, gender andthe use of anti-cancer therapy was examined by Fish-ers exact test and was not found to be statisticallysignicant. However, P. gingivalis was detected in thesamples of submandibular and submental lymphnodes of ~ 20% of these patients, and the study sug-gested that translocation of periodontopathic bacteriamay occur via lymphatic drainage, irrespective of thecancer disease status or therapy (Amodini Rajakar-una et al., 2012).In a 2005 study an increased ratio of P. gingivalis

in saliva samples of 45 OSCC patients comparedwith 229 healthy subjects was reported. Although this



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increase was not statistically signicant, this was per-haps due to the relatively small patient size (Mageret al., 2005). On the other hand, a recent analysis oforal tissue samples from 10 patients and ve healthyindividuals using anti-P. gingivalis (ATCC33277) -spe-cic immunohistochemistry detected a statistically sig-nicant increase of P. gingivalis staining in OSCCtissues than normal tissues. The study used antibodystaining against the commensal bacterium Strepto-coccus gordonii as a control, which did not show anysignicant distribution difference (Katz et al., 2011).This nding highlighted for the rst time a noteworthyassociation of P. gingivalis with OSCC, despite thelow sample size.Another study from 2012 that measured plasma

antibodies to 25 oral bacteria in pre-diagnostic bloodsamples from 405 pancreatic cancer patients and 416matched controls also indirectly associated P. gingi-valis with increased risk of pancreatic cancer. Thestudy demonstrated high correlations for two strains ofP. gingivalis (ATCC 53978 and 33277). Individualswith high levels of antibodies against P. gingivalisATTC 53978 had a two-fold higher risk of pancreaticcancer than individuals with lower levels of anti-P. gin-givalis antibodies (Michaud et al., 2012). A year laterin a clinical study involving 37 cases of chronic atro-phic gastritis, intestinal metaplasia, or dysplasia, DNAlevels of periodontopathic bacteria, including P. gingi-valis, were measured in plaque and saliva samples byreal-time quantitative PCR (Salazar et al., 2013). Anelevated but not statistically signicant odds ratio forgastric precancerous lesions was observed in relationto increasing colonization with P. gingivalis, Aggrega-tibacter actinomycetemcomitans and Treponemadenticola measured in plaque, and so the study asso-ciated high levels of periodontal-pathogen colonizationwith an increased risk of gastric precancerous lesionsin individuals with periodontal disease (Salazar et al.,2013). A recent casecontrol study examined thefecal microbiome changes of 19 colorectal cancerpatients in comparison to 20 healthy people using16S rRNA pyro-sequencing (Wu et al., 2013). Inter-estingly, although the study was not designed toscreen for specic bacterial species, it detected ahighly statistically signicant increase of the Porphy-romonadaceae family, along with Fusobacteriaceae,and three other operational taxonomic units in thecolorectal cancer patients compared with the healthysubjects (Wu et al., 2013).

All these studies underlined the plausible role ofP. gingivalis in the variety of orodigestive cancersdescribed, and prompt many questions on the possi-ble mechanistic bases of the detected associations.Answers to such questions may be obtained throughthe use of physiologically relevant in vitro studies.The rest of the review is dedicated to describing thepresently available work in the literature and willprovide further insights.

IN VITRO STUDIES AND POTENTIALMECHANISMS

Although there is mounting clinical evidence that linksP. gingivalis and/or periodontal disease with orodiges-tive cancer risk, there are currently only a few in vitroreports that directly studied the carcinogenic potentialof P. gingivalis. Based on the new information owingand the available new technologies, there is a newcharge for the scientic community to investigate theemerging role of P. gingivalis in the increased risk ofcancer development.

Findings in vitro with oral cancer cell lines

In 2011, a study used two OSCC-derived cell lines,the metastogenic SCC-25 and non-metastatic BHYcells, as well as human primary OECs, to examinethe expression of two human B7-homologue recep-tors, B7-H1 and B7-DC (Groeger et al., 2011).These receptors have been found to be highlyexpressed in the majority of human cancers. Recep-tor B7-H1, mostly known as programmed death ligand1, is an important regulator in cell-mediated immunityby promoting the development of regulatory T cells,and is thought to play a major role in suppressing theimmune system during cancer (Greaves & Gribben,2013). B7-DC, also known as programmed deathligand 2, is expressed mainly on dendritic cells andmacrophages, although it can be induced in a varietyof cell types and is suggested to suppress T-cell sur-vival, cytokine production and proliferation (Rozaliet al., 2012). In the study, Groeger and colleaguesdemonstrated a signicant upregulation of both B7-H1 and B7-DC receptors in the metastogenic SCC-25cell line and in human primary OECs after infectionwith P. gingivalis strains ATCC 33277 or W83. Incontrast, infection with Streptococcus salivarius K12,a commensal bacterium used as a control in the



study, did not have any effect on the examined recep-tors that are important for cancer etiology (Groegeret al., 2011). These ndings strongly suggest apotential role for P. gingivalis in contributing to thecellular/molecular changes promoting oral cancer,possibly through facilitating immune evasion.An earlier in vitro study examined the effect of

P. gingivalis lipopolysaccharide (LPS) on cancer pro-gression of an OSCC-derived cell line, YD-10B,which has been shown to express functional Toll-likereceptors (Park et al., 2010). The study showed noeffect of P. gingivalis LPS on the cell proliferation,migration, invasion or angiogenesis, which are themechanisms shown to be involved in the develop-ment of various cancers (Park et al., 2010). Thestudy, however, used puried LPS, and was notdesigned to study the outcome of complete P. gingi-valis infection on the cancer cell line, so excludingthe possible roles of other virulence factors of thebacterium in this model. This result may suggest thatthe reputed role of P. gingivalis is possibly not due toone singular component, but is probably a greatlyconcerted synergistic play, requiring other structuralproperties, and potentially dynamic bacteriumhostinteractions.Very recently, another study examined the effect of

P. gingivalis, using strain ATCC 33277, on themetastatic ability of two OSCC cell lines, the highlymetastogenic SAS cells and the low-metastogenicCa9-22 cells (Inaba et al., 2013). Metastatic invasionwas measured by the induction and activation ofhuman matrix metalloproteinase 9 (MMP9) in the cellsupon infection with P. gingivalis compared withanother periodontal organism Fusobacterium nuclea-tum. In SAS cells wild-type P. gingivalis ATCC 33277was able to increase basal expression of proMMP9enzyme and subsequently to activate MMP9,whereas F. nucleatum did not alter expression or acti-vation of proMMP9. In the less metastatic Ca9-22 cellline, P. gingivalis infection did not have a directlyobserved effect on proMMP9 expression or activation,although incubation of Ca9-22 cells with the superna-tant of P. gingivalis-infected SAS cells increased themetastogenic capacity of the Ca9-22 cells signi-cantly. Using gingipain mutant strains, the productionand activation of proMMP9 was shown to be depen-dent on gingipains of P. gingivalis. The study alsoshowed that proMMP9 production was controlled bythe phosphorylation of the heat-shock protein 27,

HSP27, which was previously found to be over-expressed in OSCC tissues (Turhani et al., 2006).Porphyromonas gingivalis may hold the potential tocontribute to the cancer progression and mediateenhanced host-cell metastatic potential by activationof MMP9 via its gingipain proteases (Inaba et al.,2013).Collectively, these studies substantiate the growing

plausible role of P. gingivalis in cancer etiology,rather than being a bystander, although there hasbeen a view that the opportunistic bacteria may comeafter the disease-induced suppression in immunityand compromised mucosal integrity (Cummins &Tangney, 2013). Therefore, it is important to recog-nize that studying precancerous lesions of the oralmucosa, before malignancy, is perhaps the most rele-vant stage to examine the direct involvement ofpathogens in cancer formation. The following sectionof this review will illustrate our present knowledgederived from human primary OECs and oncogenesis-related pathways, and highlight plausible mechanismsfor the role of P. gingivalis in the cellular/molecularmechanisms that are cancer promoting.

Relevant ndings with human primary oralepithelial cells

Porphyromonas gingivalishost interaction studied inprimary cultures of OECs has proven to be an invalu-able model to appreciate the highly orchestrated,complex nature of the interface between the chronicorganism and the oral epithelium, as the OECs canemulate the in vivo characteristics of human mucosalepithelium most closely. During those studies, thehuman primary OEC model provided substantialmechanistic insights into P. gingivalis persistencestrategies in the oral epithelium that may well be per-tinent in orodigestive cancer etiology and/or advance-ment.Porphyromonas gingivalis was shown to rapidly

invade the primary OECs in culture by using integrinb1 and downstream signaling pathways, and canspread intercellularly later in the infection using actincytoskeleton, so evading recognition by the immunesystem (Yilmaz, 2008). Using P. gingivalis strainATCC 33277 the organism was demonstrated to inhi-bit OEC programmed cell death induced by potentpro-apoptotic agents. The characterized epithelialhostsurvival molecular actions of P. gingivalis



include blocking of mitochondrion-dependentapoptosis (by inhibition of cytochrome c release, mito-chondrial membrane depolarization, balancing of pro-apoptotic Bax and anti-apoptotic Bcl-2 expression),activation of the phosphatidylinositol 3 kinase (PI3K)/Akt and survivin/signal transducer and activator oftranscription 3 (STAT3) survival pathways, inhibitionof caspase-3 and caspase-9 activation, and inactiva-tion of pro-apoptotic Bad-signaling (Nakhjiri et al.,2001; Mao et al., 2007; Yilmaz, 2008; Yao et al.,2010). PI3K/Akt and Survivin/STAT3 are welldescribed as key regulators of cellular processes thatcan lead to initiation and/or maintenance of carcino-genesis. Both of the pathways are currently evaluatedas major therapeutic targets in cancer (Altieri, 2003).The studies with the human primary OECs alsorevealed that P. gingivalis infection promotes a pro-survival phenotype in the host cells by acceleratingthe progression through the S-phase of the cell cyclevia the modulation of pathways involving cyclins andp53 (Kuboniwa et al., 2008; Yilmaz, 2008). The infec-tion by the organism is able to alter the expression ofp53 tumor suppressor, which is known to beinvolved in the DNA damage response and in tumorsuppression (Ozaki et al., 2013). The aforementionedactions of P. gingivalis appeared to be dependent onthe presence of the major mbriae (FimA) of theorganism (Kuboniwa et al., 2008). Furthermore,P. gingivalis can inhibit OEC cell death induced by animportant physiological pro-apoptotic molecule, extra-cellular ATP. ATP elicits its activity through the puri-nergic cell surface receptor, P2X7. The ATP-coupledP2X7 signaling has direct implications for regulationof a variety of specic host immune response ele-ments that were recently shown to be criticallyinvolved in pathways towards carcinogenesis (Roger& Pelegrin, 2011; Adinol et al., 2012; Di Virgilio,2012; Jelassi et al., 2013; Chadet et al., 2014). Someof these actions include the modulation of intracellularreactive oxygen species (ROS), controlling of apopto-sis, activation of inammasome, and secretion ofpro-inammatory cytokines. The recent efforts in thetreatment and invasiveness of several types of can-cers with high mortalities are directly targeted againstP2X7/ATP signaling, and P2X7 expression has beenfound in diverse tumors and has newly been pro-posed as a potential cancer cell biomarker (Roger &Pelegrin, 2011; Huang et al., 2013). Inhibition of ATP/P2X7-induced cell death by P. gingivalis in OECs is

mediated by secretion of an ATP-utilizing enzyme,nucleoside diphosphate kinase (NDK) (Spooner &Yilmaz, 2012; Choi et al., 2013). NDKs have recentlyemerged as a novel secreted effector for successfulpersistence by chronic opportunistic pathogens, suchas Mycobacterium tuberculosis and Pseudomonasaeruginosa (Yilmaz, 2008). A new study further dem-onstrated that P. gingivalis can modulate ATP-induced cytosolic and mitochondrial ROS as well asthe antioxidant glutathione response generatedthrough P2X7/NADPH-oxidase interaction via tempo-ral secretion of the NDK (Choi et al., 2013). ROS canserve as a key mediator in activation of a selection ofcancer/inammation-associated transcription factors(Spooner & Yilmaz, 2011).Intriguingly, NDK species, including the human ho-

mologues known as non-metastatic cell expressedproteins (NME), encoded by nm23 genes, are widelystudied for their involvement in various forms ofhuman cancers, such as breast, lung and thyroid can-cers and neuroblastoma, as well as for metastasisand regulation of p53-mediated transcription (Boissan& Lacombe, 2011; Marino et al., 2012; Prabhu et al.,2012). Most interestingly, the human NDK was shownto be over-expressed in surgically excised tumor tis-sues of patients with OSCC, compared with healthymucosa from the same patients (Turhani et al.,2006). The study identied only 20 proteins withaltered expression in OSCC tissue and intriguinglyhuman NME1 and HSP27 were among the 14proteins with signicantly increased levels.Notably, ATP/P2X7 signaling is associated with the

development of multiple pathological conditions inaddition to cancer such as diabetes, obesity, multiplesclerosis, joint disease, pancreatic disease and kid-ney diseases. The signicance of P2X7 receptorssplice and polymorphism variants in chronic humandiseases is also becoming an area of active research(Hillman et al., 2005; Lopez-Castejon et al., 2010;Chen et al., 2011; Garcia-Hernandez et al., 2011;Oyanguren-Desez et al., 2011; Adinol et al., 2012;Di Virgilio, 2012; Sun et al., 2012). P2X7 is known toplay a critical role in promoting cell growth, neovascu-larization, tumorhost interactions and metastasis,and can induce the release of cathepsins by macro-phages (involved in joint diseases) markedlyincreased in chronic and inammatory conditions (kid-ney disease, diabetes). It is worth emphasizing thatthis assembly of diseases is similar to the array of



chronic conditions recently being associated withpresence of P. gingivalis (Hitchon et al., 2010; Katzet al., 2011; Ahn et al., 2012b; Aemaimanan et al.,2013; Michaud, 2013). Moreover, molecular actions ofP. gingivalis have been shown to be highly cell-typedependent and different outcomes may be stimulatedin different cell types. These interactions are furthercomplicated by the fact that P. gingivalis colonizationin the oral mucosa is closely accompanied by multiplebacterial species. Intriguingly, among those oralorganisms, F. nucleatum is newly associated withprogression of malignant tumors of the intestinal sys-tem, particularly colorectal cancer (Kostic et al., 2013;

Rubinstein et al., 2013), thus making the entirehostpathogen interaction much more complex.Taken together, the abilities of P. gingivalis to mod-

ulate ATP/P2X7 signaling, to secrete an anti-apoptoticenzyme, NDK, during the infection of primary OECs,and to express other virulence factors (e.g. mbriae,gingipains, peptidylarginine deiminase) may serve aspotential etiological links with orodigestive cancersand possibly other chronic diseases. The postulatedstepwise transformation of the normal epithelium tothe precancerous and cancerous lesions in thepresence of P. gingivalis infection and the plausibleassociated pathways are sketched in Fig. 2.

Figure 2 Postulated pro-cancer molecular circuitry of Porphyromonas gingivalis and oral epithelial cell interface that may affect the increasedrisk of orodigestive cancers and their poor prognosis. Over the period of its co-evolution with human oral mucosa, P. gingivalis has estab-

lished multiple virulence factors such as distinctive mbriae (FimA), cysteine proteases (gingipains), lipopolysaccharide (LPS), peptidyl-argi-nine deiminase (PAD) and nucleoside diphosphate kinase (NDK). These molecules, in concert with genetic predisposition, behavioral factorsand possibly in synergy with other microbiome components, can elicit multiple pro-survival effects on the epithelial cells that may lead to pre-

disposition for the cancerous transformation. Additionally, gingipains are suggested to play a role in the activation of matrix metalloproteinases(MMPs), particularly MMP9, which are shown be associated in the metastatic dissemination of carcinoma cells. Abbreviations: IL-1b, interleu-kin-1b; NF-jB, nuclear factor-jB; PI3K, phosphatidylinositol 3 kinase; ROS, reactive oxygen species; TLR, Toll-like receptor.



Conclusion and future studies

The potential signicance of P. gingivalis is rapidly ris-ing in the OSCC etiology and the other cancers of theorodigestive tract. Porphyromonas gingivalis is thoughtto have co-evolved with humans since the divergenceof the Old World monkeys of Africa around 67 millionyears ago (Mikkelsen et al., 2008). One can speculatethat this long co-existence may have brought the excel-lent adaptation of this successful persistent bacteriumto the human oral mucosa. Cancer is considered to bea disease of civilization. It is possible that P. gingivalishas recently evolved to become a risk factor in the can-cer etiology. Conceivably, the advancements in soci-ety, the global ecological changes, and the increase inhuman longevity have allowed the increase of many ofthe current chronic diseases, and stimulated the riseof a range of currently unknown virulence factors ofP. gingivalis. Nevertheless, the aforementioned possi-bilities remain to be further researched.In summary, oral mucosa harbors vast numbers of

naturally occurring microbes on the epithelial sur-faces, which might have direct synergistic or antago-nistic effects on the occurrence and or advancementof precancerous and cancerous formations in the oralcavity. The centrality of P. gingivalis to orodigestivecancer is still debated, however, in light of the recentndings, we propose that the organism is perhaps animportant predisposing factor in the microenvironmentthat directs the development or poor prognosis of oro-digestive cancers and possibly other chronic dis-eases. Future large-scale integrated clinical andin vitro mechanistic studies accompanied by in vivovalidation assays may determine the specic micro-bial components and the associated microbiome pop-ulations that may well participate in cancer and thecomplex bacteriahost interaction contributing tothese devastating chronic diseases.

ACKNOWLEDGEMENTS

The authors would like to acknowledge the support ofthe NIDCR grant R01DE016593.

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