The Story of Human Cytomegalovirus and Cancer: = Increasing Evidence and Open Questions

Abstract

Although human=20 cytomegalovirus (HCMV) is generally not regarded to be an = oncogenic virus,=20 HCMV infection has been implicated in malignant diseases from = different=20 cancer entities. On the basis of our experimental findings, we = developed=20 the concept of =E2=80=9Concomodulation=E2=80=9D to better explain = the role of HCMV in=20 cancer. Oncomodulation means that HCMV infects tumor cells and = increases=20 their malignancy. By this concept, HCMV was proposed to be a = therapeutic=20 target in a fraction of cancer patients. However, the clinical = relevance=20 of HCMV-induced oncomodulation remains to be clarified. One = central=20 question that has to be definitively answered is if HCMV = establishes=20 persistent virus replication in tumor cells or not. In our eyes, = recent=20 clinical findings from different groups in glioblastoma patients = and=20 especially the detection of a correlation between the numbers of=20 HCMV-infected glioblastoma cells and tumor stage (malignancy) = strongly=20 increase the evidence that HCMV may exert oncomodulatory effects. = Here, we=20 summarize the currently available knowledge about the molecular = mechanisms=20 that may contribute to oncomodulation by HCMV as well as the = clinical=20 findings that suggest that a fraction of tumors from different = entities is=20 indeed infected with HCMV.

Introduction

Human cytomegalovirus (HCMV) is a ubiquitous herpes virus that = leads to=20 a life-long persistence. The frequency of infection ranges from = 50% to=20 100% in the general adult population. Human cytomegalovirus causes = severe=20 and often fatal disease in immunocompromised individuals including = recipients of organ transplants and AIDS patients. It routinely=20 reactivates in healthy virus carriers, but this is usually = controlled by=20 the host immune response [1=E2=80=933].=20 Monocytes may be an important reservoir for latent HCMV; however, = the=20 primary reservoir may be a more primitive cell from the myeloid = lineage.=20 Reactivation may result from cellular differentiation or = inflammation [1=E2=80=933].

The (possible) relationship between HCMV infection and cancer = has been=20 investigated for decades (for review see, e.g., [1]).=20 Detection of viral DNA, mRNA, and/or antigens in tumor tissues as = well as=20 seroepidemiologic evidence suggested a role of HCMV infection in = the=20 etiology of several human malignancies.

In the 1970s, the group of Fred Rapp reported HCMV to transform = normal=20 human embryonal cells in vitro [4,5].=20 Although the transformed cell lines exhibited enhanced = tumorigenicity in=20 nude mice, the expression of HCMV-specific antigens in the = transformed and=20 tumor-derived lines decreased with increasing passage [6].=20 In later studies using normal rodent cells, HCMV (infectious virus = or=20 virus DNA) was shown to induce mutations in genes that are = critical for=20 malignant transformation [7=E2=80=9310].=20 However, viral DNA was not detected in most transformants. These = findings=20 led to the speculation that HCMV contributes to oncogenesis by=20 =E2=80=9Chit-and-run=E2=80=9D mechanism [7=E2=80=9310].=20 However, this scenario is difficult to prove because it supposes = that=20 virus nucleic acids are not retained in transformed cells. In = fact, up to=20 now, there is no conclusive evidence for the transformation of = normal=20 cells after HCMV infection in humans, and the mechanism by which = HCMV=20 might contribute to oncogenesis remains obscure. Today, it is = generally=20 accepted that infection of normal permissive cells with HCMV does = not=20 result in malignant transformation (cells actively expressing = virus no=20 longer divide and eventually die), so that the virus is not = considered to=20 be oncogenic.

Twelve years ago, we proposed the concept of oncomodulation in = which=20 HCMV may favor tumor progression without being an oncogenic virus = to=20 explain the frequent presence of HCMV in tumor tissues [11].=20 Oncomodulation means that HCMV may infect tumor cells and modulate = their=20 malignant properties, in a fashion not involving direct = transformation. We=20 postulated that tumor cells provide a genetic environment, = characterized=20 by disturbances in intracellular signaling pathways, transcription = factors, and tumor suppressor proteins, that enables HCMV to exert = its=20 oncomodulatory potential, although it cannot be manifested in = normal=20 cells. To study these effects, we established persistently = HCMV-infected=20 cancer cell lines (see, e.g., [11=E2=80=9314]).=20 Studies in these cell lines demonstrated that long-term persistent = HCMV=20 infection is necessary to fully express oncomodulatory effects.=20 Subcutaneous injection of persistently HCMV-infected neuroblastoma = cells=20 resulted in increased malignant behavior as indicated by enhanced = tumor=20 growth and metastasis formation compared to noninfected cells [11].=20 Moreover, we identified HCMV as a potential therapeutic target for = patients with HCMV-infected tumors [11,13,14].

Human cytomegalovirus=E2=80=94induced oncomodulation may result = from the=20 activity of virus regulatory proteins and noncoding RNA, which = influence=20 properties of tumor cells including cell proliferation, survival,=20 invasion, production of angiogenic factors, and immunogenicity. As = a=20 result, HCMV infection may lead to a shift to a more malignant = phenotype=20 of tumor cells and tumor progression [1,14].=20 The clinical relevance of these experimental findings remains a = matter of=20 debate. We will first briefly summarize the molecular mechanisms = that may=20 underlie HCMV-induced oncomodulation before we discuss the current = evidence concerning its clinical relevance.

Molecular Mechanisms of HCMV-Induced = Oncomodulation

Influence of HCMV on the Cell = Cycle of=20 Cancer Cells

In normal permissive cells, HCMV-encoded = regulatory=20 proteins induce cell cycle arrest and prevent cellular DNA = replication but=20 maintain an active state that enables replication of viral DNA [15,16].=20 In HCMV-infected cells, the expression of the cyclins D1 and A is=20 inhibited, although hallmarks of S-phase, including pRB=20 hyperphosphorylation, cyclin E and cyclin A kinase activation, and = expression of many S-phase genes are present.

Several HCMV regulatory proteins such as the 72-kDa immediate = early-1=20 (IE1-72), 86-kDa IE2-86, and the tegument protein pp71 were shown = to=20 interact and inactivate proteins of the Rb family (pRb, p107, and = p130)=20 promoting entry into S phase of the cell cycle. Moreover, the UL97 = protein=20 was shown to exert cyclin-dependent kinase activity resulting in=20 phosphorylation and inactivation of pRb [17].=20 Conversely, HCMV IE2-86 may induce cell cycle arrest by activating = an=20 ataxia telangiectasia mutated gene=E2=80=94dependent = phosphorylation of p53 at=20 Ser15. These events result in p53 accumulation and activation, = leading to=20 a p53- and p21-dependent inhibition of cell cycle progression [18].=20 Other virus regulatory proteins, for example, the pUL69, = contribute to=20 HCMV-induced cell cycle arrest [15].

In tumor cells, cell cycle is commonly deregulated [19,20].=20 In cells with disrupted cell cycle control mechanisms (such as = tumor=20 cells), the function of virus regulatory proteins may depend on = the=20 internal context of tumor cells [14]=20 (Figure=20 1). In normal fibroblasts expressing wild-type p53, HCMV = IE1-72=20 protein cannot drive cells out of quiescence, whereas IE1-72 can = induce S=20 phase and delay cell cycle exit in p53-deficient cells. Human=20 cytomegalovirus IE2-86 protein induces a G₁/S block in = human=20 cells with wild-type pRb, but not in the human Rb-deficient = osteosarcoma=20 cell line Saos-2. In T89G glioblastoma cells with disrupted p53 = signaling,=20 persistent HCMV infection did not induce cell cycle arrest and = virus=20 antigen-positive cells continued to divide [21].=20 In experiments using a panel of human glioblastoma cell lines, a = stable=20 expression of IE1-72 was shown to differentially affect cell = growth,=20 resulting either in cell proliferation or in arrest. In U87 and = U118=20 glioblastoma cells, IE1-72=E2=80=94induced proliferation was = paralleled by=20 reduction in steady state expression levels of pRb and p53 family=20 (including p53, p63, or p73) members. In contrast, IE1-72 = expression in=20 LN229 and U251 glioblastoma cells was associated with increased = expression=20 of p53 family proteins, accompanied by growth arrest [22].=20 The HCMV protein US28 promoted cell cycle progression and cyclin = D1=20 expression in cells with a tumorigenic phenotype, whereas it = induced=20 apoptosis in nontumorigenic cells [23].

Figure=20 1

Major = effects of=20 HCMV on regulators of tumor cell cycle and/or apoptosis. = CDK,=20 cyclin-dependent kinase; E2F, E2F transcription factor; = MDM2, mouse=20 double minute = 2.

It has also been shown that persistent HCMV infection of tumor = cells=20 may lead to a selection of novel virus variants characterized by = changes=20 in coding sequences for virus regulatory proteins that have lost = their=20 ability to induce cell cycle arrest. Human cytomegalovirus = persistent=20 infection of tumor cells including glioblastoma and osteosarcoma = results=20 in the development of mutated virus variants, which grow slowly = and yield=20 lower amounts of progeny virus compared to wild-type virus strains = originally used for infection [24,25].=20 These HCMV variants were found to have DNA deletions in their = genome and=20 synthesized IE protein different in size from those of the = wildtype virus=20 strain. Moreover, stable expression of HCMV IE2-86 protein in=20 retrovirus-transduced fibroblasts did not abrogate the = G₁=20 checkpoint owing to a mutation within a critical carboxyl-terminal = domain=20 of IE2-86 protein, thus making it unable to halt cell cycle = progression=20 [26].=20 Infection of fibroblasts with an HCMV strain containing a deletion = in the=20 UL69 gene failed to induce a block in the G₁/S phase of = the=20 cell cycle [27].=20 These findings demonstrate that the effects of HCMV on cell cycle = and cell=20 proliferation may depend both on the context of the internal = cellular=20 environment and on the properties of virus regulatory = proteins.

Influence of HCMV on Cancer = Cell=20 Apoptosis

Resistance to apoptosis is a common feature of = cancer cells=20 and represents a relevant chemoresistance mechanism [19,20,28=E2=80=9330].=20 The first study on HCMV infection and apoptosis revealed that HCMV = protects fibroblasts from apoptosis induced by adenovirus E1A = protein [31].=20 Moreover, the HCMV IE1-72 and IE2-86 proteins inhibited apoptosis = induced=20 by the adenovirus E1A and TNF-=CE=B1 but not by irradiation with = UV light in=20 cervix carcinoma HeLa cells [31].=20 The authors speculated that HCMV exerts its antiapoptotic effects = through=20 IE proteins by both p53-independent and p53-dependent mechanisms. = In fact,=20 other investigators revealed that, in some cells, HCMV IE2-86 = binds to=20 p53, inhibits its transactivating function, and protects from = p53-mediated=20 apoptosis under conditions that would otherwise induce the pathway = [32=E2=80=9336]=20 (Figure=20 1). IE2-86 inhibited doxorubicin-induced apoptosis in smooth = muscle=20 cells, indicating that IE2-86 can suppress p53-mediated apoptosis = after=20 DNA damage [35].=20 Furthermore, ts13 cells with a temperature-sensitive mutation in=20 TAF_II250 do not undergo p53-dependent apoptosis when = IE2-86 is=20 expressed and the cells are grown at the nonpermissive temperature = [36].

Direct antiapoptotic activity of HCMV proteins was ascribed = mainly to=20 some distinct transcripts encoded by the HCMV UL36-UL38 genes [37,38].=20 The product of UL36, a viral inhibitor of caspase activation, = binds to the=20 prodomain of caspase-8, thus inhibiting Fas-mediated apoptosis [39,40].=20 The UL37 gene product, UL37 exon 1 (UL37x1), a viral=20 mitochondrial inhibitor of apoptosis, inhibits the recruitment of = the=20 proapoptotic endogenous Bcl-2 family member Bax and Bak to = mitochondria,=20 resulting in their functional neutralization [41,42].=20 Notably, cervix carcinoma HeLa cells expressing the viral = mitochondrial=20 inhibitor of apoptosis were resistant to apoptosis triggered by=20 doxorubicin. The HCMV UL38 gene encodes a protein that protects = infected=20 cells from apoptosis induced by a mutant adenovirus lacking the=20 antiapoptotic E1B-19K protein or by thapsigargin, which disrupts = calcium=20 homeostasis in the endoplasmic reticulum [43].=20 In cells infected with wild type virus, pUL38 was shown to = interact with=20 tuberous sclerosis tumor suppressor protein complex resulting in a = failure=20 to regulate the mammalian target of rapamycin complex 1 [44].=20 Viral 2.7-kb noncoding RNA (=CE=B22.7) inhibited apoptosis in = infected U373=20 glioma cells subjected to mitochondrial stress by stabilizing the=20 mitochondrial respiratory chain complex I [45].

Human cytomegalovirus infection was shown to protect tumor = cells from=20 apoptosis by the induction of cellular proteins, including AKT, = Bcl-2, and=20 =CE=94Np73=CE=B1 [13,14].=20 Human cytomegalovirus binding to its cellular receptors such as = integrins=20 initiates activation of AKT through the phosphatidylinositol = 3-kinase=20 (PI3K) pathway. Moreover, stable expression of the IE1-72 protein = was=20 sufficient to sustain increased AKT activity in glioblastoma cells = independently of virus receptor signaling [22].=20 Recently, HCMV glycoprotein B was shown to bind to the = platelet-derived=20 growth factor receptor (PDGFR) and to initiate activation of AKT = through=20 the PI3K pathway in different cell types including U87 glioma = cells [46]=20 (Figure=20 2). Furthermore, HCMV activated AKT by selective = phosphorylation of=20 the upstream nonreceptor cellular kinase focal adhesion kinase = (FAK) at=20 Tyr397, in glioblastoma and prostate carcinoma cell lines [47,48].=20 Activation of other signaling pathways including those mediated by = mitogen-activated protein kinase (MAPK) kinase (MEK) 1/2 or the = c-Jun=20 NH₂-terminal kinase (JNK) may be induced through viral=20 regulatory proteins and/or binding of HCMV glycoproteins to PDGFR = or virus=20 coreceptors including integrins and Toll-like receptor 2 [1,46].=20 These complex events may result in gene transcription, which = alters=20 apoptotic responses and other malignant properties of tumor cells = (Figure=20 2).

Figure=20 2

Major = signaling=20 pathways activated by HCMV binding to the PDGFR and/or by = HCMV=20 immediate early (IE) proteins that may contribute to = oncomodulation=20 by HCMV. Akt, murine thymoma viral (v-akt) oncogene = homolog-1=20 (protein kinase B); ERK, extracellular = signal-regulated=20 (more ...)

Increased expression of the antiapoptotic protein Bcl-2 and = decreased=20 sensitivity to chemotherapeutic agents were reported in = HCMV-infected=20 neuroblastoma and colon carcinoma cell lines [13,49].=20 Other observations showed that HCMV infection induced accumulation = of the=20 =CE=94Np73=CE=B1 isoform in glioblastoma and neuroblastoma cell = lines [50,51].=20 Human cytomegalovirus-induced =CE=94Np73=CE=B1 exerted a = dominant-negative effect on=20 p73=CE=B1- and p53-dependent apoptosis in both p53-negative and = p53 wild type=20 tumor cells [50]=20 (Figure=20 1). In persistently infected neuroblastoma cells, HCMV = decreased the=20 expression of p73 with a concomitant increase in N-myc=20 expression, suggesting that HCMV could also enable neuroblastoma = cells to=20 escape the apoptotic properties of p73 through = N-myc-dependent=20 pathway [50].

Influence of HCMV on Cancer = Cell=20 Invasion, Migration, and Adhesion to the Endothelium

Cancer = cell=20 invasion, migration, and adhesion to the endothelium play = important roles=20 during formation of metastases [52=E2=80=9354].=20 Neuroblastoma cells persistently infected with HCMV expressed = increased=20 motility and adhesion to human endothelial cells [1].=20 The increased adhesion to endothelium was mediated by activation = of=20 =CE=B2₁=CE=B1₅ integrin on a surface of = infected tumor cells,=20 which led to a focal disruption of endothelial cell integrity, = thus=20 facilitating tumor cell transmigration. Moreover, HCMV = downregulated=20 neural cell adhesion molecule (NCAM; CD56) receptors in = persistently=20 infected neuroblastoma cells contributing to augmented tumor cell = adhesion=20 and transendothelial penetration [51].=20 The effects of HCMV on NCAM also account for decreased adhesion of = cancer=20 cells to each other, which is presumably one of the first steps in = metastasis. The inhibitory effects of HCMV on NCAM expression may = stem=20 from the suppression of p73 expression in infected cells, = resulting in=20 decreased transactivation of the NCAM promoter by p73.

In human prostate cancer PC-3 cells, HCMV infection upregulated = tumor=20 cell adhesion to the endothelium and to extracellular matrix = proteins.=20 This process was accompanied by the enhancement of = =CE=B21-integrin surface=20 expression, elevated levels of integrin-linked kinase, and = phosphorylation=20 of FAK at Tyr397 [47].=20 In human malignant glioma cells, HCMV infection also increased=20 extracellular matrix=E2=80=94dependent migration and invasion in = dependence on FAK=20 phosphorylation at Tyr397 [48].=20 These effects were not observed in normal astroglial cells, = suggesting=20 that HCMV can selectively augment glioma invasiveness.

US28 protein, a G protein=E2=80=94coupled chemokine receptor = encoded by HCMV,=20 induces arterial smooth muscle cell migration by a = ligand-dependent=20 process [55].=20 It has been shown that US28 signals through the = non=E2=80=94receptor protein=20 tyrosine kinases Src and FAK and that this activity is necessary = for=20 US28-mediated smooth muscle cell migration [56].=20 It is of interest to show whether the US28 pathway may contribute = to FAK=20 activation and stimulation of cell invasion observed in = HCMV-infected=20 tumor cells.

Investigation of gene expression in several persistently = HCMV-infected=20 neuroblastoma cell lines by gene microarray revealed up-regulation = of=20 genes that are involved in cancer cell invasion [57].

Influence of HCMV on=20 Angiogenesis

Recruitment of tumor vessels is an integral part = of=20 cancer initiation and progression [19,58,59].=20 US28 protein, a G protein=E2=80=94coupled chemokine receptor = encoded by HCMV,=20 induced a proangiogenic and transformed phenotype in mouse = fibroblast=20 NIH-3T3 cells through up-regulation of vascular endothelial factor = [23].=20 US28-expressing cells were tumorigenic in nude mice. Expression of = a G=20 protein=E2=80=94uncoupled constitutively inactive mutant of US28 = delayed and=20 attenuated tumor formation, indicating a role of constitutive = receptor=20 activity in the onset of tumor development. Importantly, US28 was = also=20 shown to be involved in HCMV-induced angiogenesis in glioblastoma = cells=20 infected with a clinical HCMV strain through the induction of = vascular=20 endothelial factor production. Moreover, expression of interleukin = 8=20 (IL-8), another well-recognized promoter of tumor angiogenesis, = was=20 stimulated by HCMV infection in leukemia and glioma cells [60,61].=20 Human cytomegalovirus-induced IL-8 expression may be caused by the = ability=20 of the HCMV IE1-72 protein to transactivate the IL-8 promoter = through the=20 cellular transcription factors NF-=CE=BAB and AP-1 [61].=20 In addition, HCMV infection of glioma cells suppressed the = expression of=20 angiogenesis inhibitors, such as thrombospondins 1 and 2, through = the=20 activity of IE proteins without the involvement of p53 [62,63].

Human cytomegalovirus infection of endothelial cells resulted = in=20 proangiogenic effects mediated through virus binding to and = signaling=20 through integrin =CE=B21, integrin =CE=B23, and epidermal growth = factor receptor [64].=20 Secretome analysis of HCMV-infected cells revealed enhanced levels = of=20 proangiogenic molecules as well as increased proangiogenic = activity of=20 cell-free supernatants [65].=20 By this mechanism, tumor cells themselves as well as = nontransformed stroma=20 cells (e.g., fibroblasts or endothelial cells) may contribute to = cancer=20 progression.

Influence of HCMV on Cancer Cell=20 Immunogenicity

The ability to evade from recognition by the immune system is = essential=20 for cancer cells [66,67].=20 The HCMV proteins US2, US3, US6, and US11 decrease cell surface = expression=20 of major histocompatibility complex (MHC) class I or class II = proteins,=20 which may help infected cells to avoid adaptive immune response = [68,69]=20 (Figure=20 3A). The HCMV-encoded protein UL16 provides = protection=20 against detection by immune cells through preventing the MHC class = I=E2=80=94related chain B, UL16 binding proteins 1 and 2 from = reaching the cell=20 surface, whereas HCMV microRNA (miR-UL112) prevents expression of = MHC=20 class I-related chain B in infected cells [68,70,71].=20 MHC class I=E2=80=94related chain B and UL16 binding proteins are = cellular ligands=20 for the activating receptor NKG2D, which is expressed on some = natural=20 killer (NK) cells, =CE=B3/=CF=83 T cells, and CD8⁺ = cells. Other=20 HCMV-encoded proteins such as MHC class I homolog UL18 and UL40 = may=20 inhibit NK responses against infected cells by triggering NK = inhibitory=20 CD94/NKG2A receptor [71]=20 (Figure=20 3B). The potential contribution of such = oncomodulatory=20 mechanisms to tumor progression was shown in a murine = cytomegalovirus=20 (MCMV) model. Expression of the MCMV-encoded class I homolog m144=20 protected lymphoma cells from NK cell lysis resulting in increased = tumor=20 growth and decreased survival in a syngeneic mouse model [72].

Figure=20 3

Immune = escape=20 mechanisms mediated by HCMV in tumor cells. (A) Influence of = HCMV=20 gene products cytotoxic T-cell lysis in tumor cells, (B) = influence=20 of HCMV gene products (and microRNA) on natural killer cell = lysis in=20 tumor cells. MHC, major histocompatibility=20 (more ...)

Human cytomegalovirus-infected tumor cells may avoid immune = responses=20 also by production of immunosuppressive cytokines. The = HCMV-encoded viral=20 IL-10 homolog (UL111a; cmvIL-10) exerts potent immunosuppressive=20 properties similar to those of IL-10 produced by human cells [73=E2=80=9375].=20 During latent infection, the UL111a region transcript undergoes=20 alternative splicing, which results in the expression of=20 latency-associated cmvIL-10. The latency-associated cmvIL-10 = retains some,=20 but not all, of the immunosuppressive functions of cmvIL-10 and = its=20 expression may enable HCMV to avoid immune recognition and = clearance=20 during latency [75].=20 This may be of relevance for tumor cells with limited = permissiveness for=20 HCMV. Moreover, HCMV stimulated the production of the cellular = immune=20 suppressive cytokine transforming growth factor =CE=B21 = (TGF-=CE=B21) in different=20 tumor cell types including glioblastoma, leukemia, and = osteosarcoma cells=20 [76,77].=20 Interestingly, TGF-=CE=B21 is regarded to be the most prominent=20 glioblastoma-associated immunosuppressant [78].=20 Moreover, TGF-=CE=B21 itself had already been shown to stimulate = HCMV=20 replication in cultured cells [79].=20 A comparative study of the influence of HCMV infection and IE = protein=20 expression on TGF-=CE=B21 promoter function in permissive cells = pointed to a=20 possible cooperative role between IE proteins and protein(s) = expressed=20 during the early phase of viral infection [76].=20 In the human glioma cell line U373, the HCMV IE2-86 protein = regulates=20 transcription of the TGF-=CE=B21 gene by its ability to interact = with the Egr-1=20 DNA-binding protein [80].=20 In addition HCMV was shown to induce integrin = =CE=B1_v=CE=B2₆=20 expression in endothelial cells of different tissues in patients = with HCMV=20 infection. The expression of integrin = =CE=B1_v=CE=B2₆ in=20 HCMV-infected cells promoted activation of TGF-=CE=B21 from its = secreted=20 biologically inactive form [81].=20 Therefore, HCMV may influence individual infected cells, = surrounding=20 tissues, and/or immune reactions through TGF-=CE=B21 production = and/or=20 activation. This may promote virus replication and interfere with = host=20 immune responses against tumor cells.

Influence of HCMV on Chromosome=20 Stability

Human cytomegalovirus infection has been demonstrated to induce = chromosome damage [82],=20 and genetic instability is considered to be major driver of cancer = progression [83,84].=20 In 1972, chromosome damage induced by HCMV infection in human = cells was=20 reported for the first time [85].=20 Human cytomegalovirus infection in combination with cytotoxic = agents=20 synergistically increased genotoxic effects [86,87].=20 Most notably, HCMV was shown to induce specific chromosome 1 = strand breaks=20 at positions 1q42 and 1q21 in a replication-independent manner [88].=20 The possible targets residing near 1q42 include the ADPRT locus = involved=20 in DNA repair and replication [89]=20 whose deletion has been connected to the development of = glioblastoma [90].=20 A breast cancer tumor suppressor gene was proposed to be located = at=20 1q21-31 [91],=20 therefore representing a potential target of 1q21 strand = breaks.

Human cytomegalovirus IE1-72 and IE2-86 gene products can = cooperate=20 with the adenovirus E1A protein to transform primary baby rat = kidney (BRK)=20 cells [9].=20 The finding that many of the transformed BRK cell lines contained = mutated=20 p53 alleles suggest that mutation of p53 might be one of the = mechanisms by=20 which IE proteins contribute to transformation. Because HCMV = proteins and=20 DNA were not present in cell lines derived from the transformed = BRK foci,=20 it has been suggested that HCMV could contribute to oncogenesis by = =E2=80=9Chit-and-run=E2=80=9D mechanism [9].=20 In addition, three different cell lines transformed by HCMV were = shown to=20 harbor an activating mutation in both alleles in H-Ras [8].=20 However, in both cases, it is unclear whether the mutations in = H-Ras or=20 p53 are a direct result of the mutagenic activity of HCMV gene = products.=20 The mutations could arise as the transformants are selected for = growth in=20 culture.

Clinical Findings

Although HCMV infection of tumor cells was initially reported = 30 years=20 ago in patients with carcinomas such as prostate or colon cancer, = later=20 pathologic investigations provided conflicting results [14].=20 A renewed interest in the role of HCMV in cancer diseases was = promoted by=20 recent studies using highly sensitive techniques for virus = detection which=20 indicated the presence of genome and antigens, of HCMV in tumor = cells (but=20 not in adjacent normal tissue) of more than 90% of patients with = certain=20 malignancies, such as colon cancer, malignant glioma, prostate = carcinoma,=20 and breast cancer [3,49,92=E2=80=9396].=20 These pathologic observations demonstrated that HCMV causes = low-grade=20 infections in tumor cells probably sustained by persistent virus=20 replication. To further understand whether HCMV infection in = tumors is of=20 clinical relevance, patients with malignant glioblastoma were = grouped=20 according to the level of HCMV-infected tumor cells. Remarkably, = patients=20 with low levels lived almost twice as long as patients with high = levels=20 suggesting that HCMV infection of tumor cells alters the disease = course in=20 this patient group [3].=20 Moreover, detection of HCMV in different histologic types of = gliomas=20 revealed that HCMV-positive cells in glioblastoma multiforme were = 79%=20 compared to 48% in lower grade tumors [96].=20 Recent clinical studies also demonstrated the presence of HCMV DNA = in the=20 peripheral blood of a high percentage of glioblastoma patients = (80%) but=20 not in the blood of healthy control individuals [94].=20 These results suggest either a systemic reactivation of HCMV = within=20 patients with glioblastoma (which may be relevant for virus = transport from=20 periphery into the tumor tissues) or shedding of viral DNA after=20 reactivation of latent HCMV in tumor cells into the periphery. = Although=20 HCMV viremia exerts subclinical character in glioblastoma = patients, it may=20 be relevant for the transport of HCMV into tumor tissues by immune = cells=20 such as monocytes/macrophages. A secondary reactivation of virus = may be=20 caused by cancer-related and/or treatment-related = immunosuppression. In=20 fact, a pilot clinical study showed that the incidence of HCMV=20 reactivation in patients receiving conventional chemotherapy = (without=20 major immunosuppressive agents) may be high without obvious HCMV = disease=20 [94].=20 It should also be noted that HCMV reactivation seems to be = dependent on=20 differentiation of myeloid lineage and inflammation [2,3].=20 In patients with different inflammatory disorders, HCMV = reactivation was=20 evident in inflamed tissues but not in non-inflamed tissue = specimens from=20 the same patient or healthy controls [3].=20 Therefore, inflammatory environment present in most solid tumors = could=20 contribute to local HCMV reactivation. Conversely, HCMV also = exerts=20 proinflammatory potential mainly owing to the production of = numerous=20 inflammatory mediators from infected cells [14].=20 Thus, HCMV reactivation in cancer tissues may enhance tumor = inflammation=20 and accelerate a malignant process.

Conclusions

Many clinical and experimental findings suggest a contribution = of HCMV=20 to malignancy and chemoresistance of infected tumor cells from = different=20 entities. However, oncomodulation needs to be further defined in a = systematic manner to increase the understanding of the phenomenon = and to=20 better translate the experimental results in more effective = anticancer=20 therapies. First, standardization of highly sensitive techniques = is=20 necessary to detect low-grade HCMV infection of tumor tissues to=20 reasonably compare pathologic studies from different groups. = Moreover, the=20 clinical relevance of experimentally defined oncomodulatory = mechanisms=20 induced by HCMV regulatory proteins and noncoding RNA needs to be=20 examined. This includes the investigation of the HCMV = oncomodulatory=20 activity in the context of the internal cellular environment in = tumor=20 tissues and genetic and functional studies of HCMV strains = isolated from=20 patients' tumor samples. Hereby, it is important to study = HCMV-induced=20 oncomodulation not only in infected tumor cells but also in stroma = cells,=20 because HCMV-induced changes in the tumor microenvironment may = also=20 contribute to oncomodulation. Another central aim is to develop=20 therapeutic strategies to suppress HCMV replication or to target = viral=20 regulatory proteins or noncoding RNA because persistent virus = replication=20 is supposed to be essential for oncomodulation. Clinical trials to = evaluate the efficacy of antiviral treatment or HCMV-targeted=20 immunotherapy in patients with malignant glioblastoma have just = been=20 started [3,94].=20 Most recently, an HCMV-specific CD8⁺ T-cell response = was=20 induced in a glioblastoma patient after therapeutic vaccination = with=20 dendritic cells pulsed with autologous tumor lysate [95].=20 This finding supports that HCMV may be a potential = immunotherapeutic=20 target in HCMV-infected tumors and should therefore be a further = impulse=20 to strengthen our effor.