Evidence for the association between Mfvmfrvs and prostate cancer 41 The Interferon IFN pathway

Evidence presented so far indicate that 1. in prostate carcinoma, an interferon-sensitive pathway appears to be affected. Attempts to identify an infectious agent (also on the basis of these observations), had led to identification of XMRV, a candidate virus, which has been eventually falsified by several groups (see part 1). However, as it has been emphasized in this chapter, evidence for viral involvement in PCa are rather strong and independent from the particular isolate XMRV. Indeed, as previously underlined, XMRV isolation is based upon usage of viro-chip technology and logical inference analysis predicts that this step is most error-prone [1]. In order to list and underline numerous elements indicating MFV as a strong candidate, the general IFN pathway is here considered and RNase-L as next point.

Although RNase-L is also an essential part of IFN pathway, it will be discussed separately, since it is prominent in view of numerous evidence and studies performed in PCa and other pathologies. Furthermore, the fact that transgenic animals knockouts for RNASEL gene do not develop tumours at higher frequency, suggests that additional elements in the IFN pathway may also be relevant [56] [57]. Since several years ago, the IFN pathways has been extensively dissected: beside RNase-L, two additional pathways are prominent: a) the PKR signal transduction and b) the Adenosine-Deaminase of RNA (ADAR) mechanism.

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Figure 8. Tumorigenesis in nude-mice of SK-N-SH cells infected/transformed by MFV. Injection of 107 SK-N-SH cells (right flank) or SH-10 clone (left flank) with the same number of MFV-transformed SK-N-SH cells shows the outgrowth 3 weeks later of large tumoral masses in the case of MFV-infected/transformed cells (SH-10). Histological analysis confirmed the presence of human neuroblastoma cells after xenotransplantation [53].

Figure 8. Tumorigenesis in nude-mice of SK-N-SH cells infected/transformed by MFV. Injection of 107 SK-N-SH cells (right flank) or SH-10 clone (left flank) with the same number of MFV-transformed SK-N-SH cells shows the outgrowth 3 weeks later of large tumoral masses in the case of MFV-infected/transformed cells (SH-10). Histological analysis confirmed the presence of human neuroblastoma cells after xenotransplantation [53].

PKR was one of the best characterized pathways in the IFN signal transduction, starting from pioneering work of Isaacs and Lindenman, who initially characterized the IFN activity

[58] [59]. One of the first enzymatic activities induced by IFN and its inducers (i.e., dsRNAs) is the dsRNA dependent Protein Kinase or PKR. PKR conveys the IFN message in several ways but especially by phosphorylation of: 1. PKR itself (autocatalysis); 2. the a subunit of eIF-2a ; 3. the inhibitor of transcription factor NFkB, IkB, thus releasing such inhibition; 4. the TAT transcription factor, essential activator of HIV; 5. the NFAT protein and 6. the phos-phoprotein MPP4, which binds dsRNA and is activated during the M phase of cell cycle [58]

[59] [60]. Among the different activities elicited by PKR activation, the best studied and known is certainly the inhibitory effect on protein synthesis (eIF-2a) [61]. Ser-51 Phosphory-lation in this case blocks initiation of protein synthesis, by inhibiting the exchange of gua-nine nucleotide [61] [62] [63]. There are some discussions and discrepancies on the regulation of PKR from different portions of the gene/structure: two knockout transgenic mice were generated by the groups of Karomillas/Bell [64] and Ch. Weissmann [65] [66].

The first one has been targeted in the carboxy-terminal of PKR, where is present the kinase activity, and doesn't show impairment of antiviral response or TNF-a responses, thus indicating the redundant role of PK activity [60, 64]. To the contrary, Weissmann's lab ko mouse and its MEFs are strongly inhibited in view of the deletion of the NH2- portion of the protein, where the dsRNA-binding domain of PKR resides [65] [66] [60]. Effectors of PKR are several types of dsRNA molecules, both artificial and natural [67]. In this respect, the genome of MFV is a strong/ideal inducer of PKR in acutely infected cells, as we have documented in both mouse and human cells. Most likely, PKR induction also contributes to the strong apoptotic effects we have documented 36-72 hours post-infection [53]. In particular, MFV-infected cells completely block protein synthesis and strongly impair rRNA production (see later) and these effects seem to be mediated by PKR and RNase-L respectively. In prostate cancer, the same pathway of PKR appears to be downstream of another essential regulator and also Tumor Suppressor function of prostate cells: PTEN [68]. Therefore, typically the deletion of PTEN (which is extremely common in prostate cancer [69]) will lead to ablation of the TSG function of PKR by phosphorylation of eIF-2a and block of protein synthesis [68] [69]. In view of the MFV/MFRV connection hypothesis, it is speculated that infection by this family of viruses will eventually cause/select for PTEN deletion, as this will inhibit cellular apoptosis, which would otherwise be inescapable [53] [55] [69].

The additional and last form of IFN response here considered is the RNA-specific Adeno-sine Deaminase or ADAR, which is strongly induced after viral infection [70] [71]. Although such RNA editing was initially considered a rare phenomenon, almost a curiosity of RNA regulation and fine tuning, extensive genomic sequencing by NGS and high-throughput technologies have allowed to discern considerable editing in several DNA genomic sequences by a related Deaminase activity which targets DNA, called APOBEC, as well as ADAR activity on expressed mRNAs [72] [73] [74]. It is still not clear how efficient such mechanism may be at the RNA level, since ADAR activity may protect cells but also favour virus aggression or persistence inside infected cells [70]. DNA deaminase activity as well is still poorly understood, but new phenomena discovered in human cancer cells through Next Generation Sequencing (NGS) technology suggest that genome modulation and plasticity by APOBEC could also play a major role in carcinogenesis [73] [75].

Could the ADAR activity induced by Interferon (ADAR-1) be responsible for important antiviral effects in human and other cells ? Although the question is still open and there are also examples of opposite regulation as previously mentioned -for example in Hepatitis Delta Virus (HDV) [70]-, important inhibitory effects were documented, with Measles Virus [76] [77] and with Influenza Virus in mouse cells [78]. Furthermore, a specific gene of the Adenovirus genome is responsible for counteracting the RNA-editing activity of ADAR: the VAI gene [79]. In the case of Reoviridae, little is still known but there is at least one animal model in which ADAR was induced by both artificial dsRNA and reoviridae genome

(which obviously is dsRNA), although the response modality of ADAR appeared quite different [80] [81].

Among human cancer as well very little is known, particularly in the case of prostate carcinoma [82] [73]. More data have been obtained in the case of brain tumours, since ADAR is known to effect important editing in brain neurons. In at least two tumour examples, hypoe-diting seems to characterize cancer cells. In glioblastoma multiforme, rectification of a mutated permeable glutamate receptor to Ca++-impermeable receptor suppressed proliferation [83] [84], while in hypo-editing astrocytoma cells, re-balancing editing expression induced regression [85]. Additional work on ADAR/APOBEC in prostatic cancer and reoviridae is certainly warranted.

4.2. RNase-L, an essential pathway

The clearest evidence for viral involvement in prostate carcinoma and in human cancer in general was obtained through studies of the IFN response leading to RNase-L activation. The general scheme of IFN genes activation has been clarified through years of intense studies with several models, cell lines, laboratory and transgenic animals, in vitro assays and molecular/biochemical systems [60] [86]. Without getting into too many details -and referring instead the readers to some excellent review articles on this subject [60] [86] [87] [88] -two general types of IFN molecules are known: viral IFN and immune IFN. Both IFN-a and IFN- p (as well as IFN-omega) belong to the viral form, induced by viral infection [60], while the immune form is essentially composed by IFN-y and is induced by immune stimulation. Focusing on viral genes, this is a rather large family in humans, since 13 genes for IFN-a, 1 for IFN-p and one for IFN-omega were mapped on the short arm of human chromosome 9 [89] [90]. None of these genes contains introns, while the only IFN gene with introns is the IFN-y form with 3, on the long arm of chromosome 12. Two types of IFN receptors, IF-NAR-1 and IFNAR-2 are known on human chromosome 21: they must heterodimerize for activation by IFN-a/p [91] [92] [93]. The following signalling is today understood mostly in the JAK-STAT transduction pathway, thanks to the work of several molecular biologists, first and foremost the group of Jim Darnell at Rockefeller University [94] [95] [96]. STATs are "signal transducers and activator of transcription" molecules: at least seven of them are known, i.e. Stat-1, Stat-2, Stat-3, Stat-4, Stat-5a, Stat-5b and Sta-6. STATs are activated by members of the Janus family of Tyrosine kinases (JAKs), of which 4 members are known, i.e. Jak-1, Jak-2, Jak-3 and Tyk-2. Various combinations of Jak's and Stat's elements are active in transducing both viral and immune IFN signalling [87] [88]. Additional important elements are the IRFs, (IFN Regulatory Factor) family [97], which cooperate in the activation of IFN-responsive genes. These are characterized by presence of regulatory elements: ISRE or IFN-stimulated Response elements [98], usually for viral IFNs and the GAS (gamma IFN activation sites) [99]. In conjunction with Stat's, IRFs constitute the so called ISGF (IFN stimulated gene factor's) [100]. Having clarified this terminology, ds-RNA activation of IFN pathway does not always or only use Jak-Stat elements. The transcription factor IRF-3 (IFN response Factor 3) acts as subunit of a complex called ds-RNA activated transcription factor complex (DRAF) by sereine/threonine phosphorylation, translocation to the nucleus, associ ation with p300/CBP and gene activation [101]. Among the activated genes, the Oligo Ade-nylate Synthetase gene family, or OAS, is one of the most important, because it conveys the anti-viral signal to the next and final effector: RNase-L. In humans, three OAS genes of different sizes (proteins of 40-46 kDa -OAS1-, of 69 kDa -OAS2- and of 100 kDa -OAS3-) have been mapped on the long arm of chromosome 12 (12q24.2), thus suggesting genome duplications in H. sapiens [102]. Similarly to the effectors previously considered -PKR and ADAR-, OAS proteins do contain regions for binding dsRNA, the signalling and activating effector [103] [104] [105]. However, all three effectors contain separate regions with peculiar enzymatic activities: kinase, deaminase, synthethase [60]. The exact nature of dsRNA activators is not always completely clear, but hypothesized to be formed by or to contain dsRNA elements. In the case of interest, i.e. MFV/MFRVs infection, since these belong to Reoviridae family, it is clearly fragments or segments of viral genome (dsRNA) [53] [55]. Great variation has been documented in the extent/level of OAS activation and 2-5 A production [80].

Last element, RNase-L, is activated by 2-5 A signal molecules, typically oligomers with >2 elements for optimal induction, while RNase-L must dimerize for activation [106] [107]. This endoribonuclease is typically present as monomer in a latent form, essentially in every cell (tested so far), but -after 2-5 A interaction- it homodimerizes and is activated [107], although heterodimers with RLI (RNase-L inhibitor) have been also described. As previously mentioned, RNase-L gene, called RNS4, has been mapped on the long arm of chromosome 1 (1q25), on a location corresponding to the chromosomal site for the Human Prostate Carcinoma susceptibility (HPC-1) [108], as mapped with linkage studies by Jeff Trent and others [108] [6].

In view of the coincident chromosomal location of RNASEL and HPC-1 [6] [108] and the initial speculations by Lengyel and others that this gene may behave as a bona fide Tumor Suppressor [109] [12] [13], Silverman with DeRisi and Ganem undertook the described viro-chip high-throughput search for potential viral candidates, leading to XMRV isolation [15]. As previously described and discussed in Section 1, XMRV identification has been clearly falsified as a recombination artefact arising during xenograft transplantation in nude mice [45].

In view of the coincidental chromosomal localization of HPC-1 and RNASEL, what are the evidence for RNase-L involvement after MFV/MFRV infection ? The acute phase of infection by these viruses is accompanied by a very high activation of RNase-L [53]. An assay, detecting ribosomal RNA (rRNA) degradation in infected and transforming cells was developed (U. Rovigatti, unpublished), thus confirming the extremely high levels of RNase-L induction, often leading to block of cell proliferation and apoptosis [53] [55]. In the past several years, groups in USA, France and Belgium have also documented a strong deregulation of this endoribonuclease in patients affectd by Chronic Fatigue Syndrome. Pioneering work by Suhadolnick et al at Temple Univ. initially disclosed that 2-5 A activated RNase-L is upregu-lated in CFS patients [110] [111] [112]. This finding was followed up by description of a lower molecular weight form (37 kDa) of the same enzyme in CFS patients by the same group [113]. The French-Belgian group of De Meirleir et al. then showed that the 37 kDa fragment is proteolytically cleaved from the original enzyme of 87 kDa, by human elastase and/or cal-

pain [114]. The same authors also speculated that the levels of 2-5 A molecules with structures larger than dimers (trimers/tetramers) protect the 83 kD moiety from degradation [115] [116]. Presence of the 37 kD RNase-L could also explain the higher enzymatic activity (associated with the low MW form) and the ratio between the two forms was proposed as potential marker for CFS [117] [111] [118].

In conclusion, the extremely elevated levels of RNase-L in every cell type infected by MFV indicate that this could be an important parameter to be evaluated. Analysis is being performed for addressing the question of whether cells with impaired/mutated alleles of RNase-L (such as the R462Q allele) may be more resistant to the apoptogenic effect(s) of MFV/MFRV and could become better targets for carcinogenesis [53] [55] [1].

4.3. Inflammation - ubiquitous in PCa

Inflammation has been estimated as being somehow responsible for 20% of human cancers: these are typically linked to infectious agents, causing chronic infections as well as by other environmental factors [119] [120] [121]. While it appears to be an essential component of carcinogenesis -being defined as "the seventh hallmark of cancer" [122]- inflammatory proceses are particularly prominent in PCa [121] [119]. Furthermore, inflammation in PCa adds an enigmatic component, which could be or become one of the best clue for deciphering its aetiology [55]. This enigmatic nature is however rather complex, as it can be distinguished by several elements:

1. The paradox of a rather common disease (most common cancer among men), afflicting this year over 300.000 people and killing more than 33.000 patients, only in the US [123]. For comparison, it has been observed that there is just a handful of cases described in the literature for Primary Seminal Vesicle Carcinoma, essentially in the same anatomical location [124]. This shows a peculiar and striking difference between two very close histological sites: PCa is diagnosed only in the prostate peripheral zone, rarely in the transition zone and almost never in the central zone [125]. This pattern is accompanied by typical phenomena of inflammation which is almost never of acute type (at time of diagnosis) and is characterized in the described zones by: a. chronic inflammation, b. benign prostatic hyperplasia (BPH), d. focal atrophy, e. a new type of inflammatory response defined as prostatic inflammatory atrophy (PIA) and finally developing into f. prostatic intraepithelial neoplasia (PIN) and/or g. prostatic carcinoma (PCa) [125] [126] [127] [128]. This pattern associates with an extremely common disease, the most common form of cancer in men, again suggesting a rather "common" causality [55]. As we will also consider other clues (see 2,3,4), the best explicatory mechanism is that of a common infection, in particular an infectious agent which is "endogenous" or "persi-tent" in H. sapiens and apparently much more frequent in certain human populations or races [129] [130] (see below)

2. Variation in epidemiological data for Chinese, Japanese or Arab men in comparison with the male population in Western Countries: for example, men born in South East Asia who then migrated to the US acquire a higher incidence within the first two gener ations [130] [131] [132]. In Shri-Lanka men, the incidence was recently assessed as 5.7 per 100.000 males, while such incidence rose up by twenty folds in immigrants to the Western Countries (for example, UK) [133]. This is again indicative of "life-style" rather than genetic factors being responsible for prostate carcinogenesis. Similar data are also present in Japanese men who migrate to the US [134]. Using similar epidemiological approaches, Hsing has recently divided different nations into three risk groups for incidence and mortality of PCa. The high risk group includes USA and several European Countries; to medium risk group belong European Nations such as UK, Italy and Spain, while Asian Countries are mostly included in the third, low risk group [130]. There is a general trend of increasing incidence, which the authors attribute to westernisation of life-style in the low-risk nations, but to TUR (trans-uretral resection) and PSA-testing in the high risk Nations [130, 134]. Interestingly, the incidence in US black males is 50-60 fold higher that that in Shanghai, China [130] [135]. The data by Julian Peto and others have confirmed these trends and the rapid changes in incidence/mortality in migrant populations (often within the first generation), thus emphasizing the concept that factors different from genetics (i.e. environmental, such as infectious agents) may be responsible [129].

3. The fact that inflammation, BPH and PCa typically occur in the prostate peripheral area, with almost no tumours in the central zone. This led initially LM Franks to hypothesize that in prostate cancer, the inflammatory effects are always accompanied by hyperproliferation and/or atrophy/necrosis [126]. Later, McNeal et al. have elaborated the same concept, by noticing in 1988 that in the whole gland, there is a clear-cut zonal distribution [125]. Out of 88 tumours studied, the majority (68%) arose in the peripheral gland, while 24% in the transitional zone and only 8% in the central gland zone. As mentioned, this suggests that infection through ascending urethra could be a responsible/associated factor [125] [136] [119]. Although bacteria have been initially suspected as responsible for inflammatory phenomena and as causes of carcinogenesis (Neisseria Gonorrhoeae, Clamydia Trachomatis, Trichomonas Vaginalis, and Treponema Pallidum), such intepretation has been reconsidered in post-antibiotics era, since prostatic persistant bacterial infections have dramatically dropped [137]. Still, bacteria can be often grown even from expressed fluids of asymptomatic men [138]. The possible causality by viruses is still an open question, as extensively discussed in the first section on XMRV discovery and refusal. Among viruses, several have been extensively investigated throughout the years and particularly: i. Cytomegalovirus (CMV) has been investigated in view of its association with malignant transformation in vitro. Seven studies on tissues and 2 on serology do not support an association with PCa. [139] [140, 141] ii.Epsten Barr Virus (EBV) levels were shown to be not significantly different in PCa/BPH by Ab's, PCR and IHC; [142] iii. HHV-8 was initially detected by Chang and Moore in Kaposi Sarcoma (KS) by subtractive hybridisation [143]. Initial positive results in PCa by Monini et al. [144] were later explained by infiltration with lymphocytes, most likely in HIV-1 positive (and therefore HHV-8+) patients [145, 146]. Also, the strongly positive findings by Hoffman in men from Trinidad and Tobago can be probably explained by bias of selected controls [147] [148]. iv. Polyoma Viruses have been also associated with PCa: an ini tial report by Monini et al. [149] was followed by an interesting paper by Das et al., since they found BKV DNA positivity more frequently in malignant tissue [150]. However, also these studies were not confirmed [151] [152]. v. Human Papilloma Viruses (HPVs) : Extensive work has focused on these viruses throughout the years. Their relevance was also surmised from studies on women cervical/uterine cancer -pioneered by Harald zur Hausen (Nobel Prize for Medicine in 2008)- where HPVs are clearly involved in >90% of cases [153]. However: i) even Zur Hausen in reviewing this subject in his Nobel lecture dismisses the role of HPVs in PCa: in this sense, men would be some kind of "healthy carriers" of the viral carcinogen [154]; ii) Several studies have been published, some of which with positive results (for example, in an Argentinean study, 42% PCa were positive versus 0% BPH samples) [155]. However, in 24 studies from other Countries, there is no evidence of different HPV involvement between cancer specimens and controls [156]. Furthermore, the most recent meta-analysis didn't show significant OR for PCa associated with HPV-16 (OR = 1.09) or with HPV-18 (OR = 1.08) infections [157]. Similar results were obtained by a recent review article (in press) [156]. iii) Another element which does not fit HPVs as potential carcinogens in PCa is the observation -initially made by Woodsworth [158] - that HPV infections do not elicit high inflammation or inflammation at all ([158], see also later discussion). As already mentioned, inflammation -most likely associated with a prostatic infection- is one of the Hallmarks of Cancer, particularly in this tumor type, PCa [122] [121].

4. Several reports (Platz; Mahmoud; Chan; Jacobs) have documented that assumption of aspirin or Non-Steroidal Anti-Inflammatory Drugs (NSAIDs) for different periods could considerably lower PCa risk [159] [160] [161] [162]. These studies have been expanded in recent years, particularly by the work of Mahmud with several Meta-Analy-ses [163] [164, 165] [166] [167]. A positive correlation has been detected for aspirin usage (protection) with OR in the range of 0.81- 0.83 and this is confirming what was already known from animal studies, which typically display stronger and clear-cut protective effects (also with NSAID). In the case of NSAID, the effect is less apparent, or maybe diluted out [166]. In most recent nested case-control studies, Mahmud has confirmed a modest but significant effect with all propionates, ie. Ibuprofen, Naproxen etc., but not with other NSAIDs [167]. The question is somehow connected also to the relationship between BPH and PCa, since the former has been often considered a precursor and initial inflammatory response leading to the latter [168] [169] [170]. Additional findings hower do not lend support to this hypothesis [171] [172]. Finally, a very recent study by Sutcliffe and others also does not show any effect for NSAID treatment in BPH as well as LUTS (Low Urinary Tract Symptoms) [173]. In either cases and also in view of the Mahmud meta-analyses, there may be a positive (protective) effect, but too small and/or diluted out [173].

4.3.1. How can this Inflammation-Scenario fit the proposed role of MFV/MFRVs a. First of all, this family of viruses infects the human population in the first years of life. By age 5, >95% of human population displays antibodies against Reoviridae and this type of viruses have been shown to be capable of persisting in infected patients and animals for several months/years [174]. Furthermore and as discussed in the next section, MFV/MFRVs display all the features of a "Stem Cell Virus" (SCV), with features of interaction in early childhood with a developing immune system [53, 175]. That a prostate cancer stem cell may be present in PCa and targeted in the first phase of carcinogenesis has been longly hypothesized and recently confirmed [176] [177] [178]. Further studies are certainly warranted in order to assess presence of MFV/MFRVs in early childhood and during ontogeny [55].

b. Different levels/types of MFV/MFRVs appear to be present in different human populations worldwide. However, a clear picture of the specific subtypes involved is still missing, particularly for what concerns MFV/MFRVs. This should be clarified experimentally (by viral nucleic acid and specific protein detection, presence of antibodies etc.) [174]. Furthermore, essential aspects of these viruses features are still missing as we do not have full knowledge of these viruses genome sequence/structure [53]. Patterns of infections and micro/mini-epidemics in different populations could be deduced and mirrored by what is happening with Rotaviruses (another member of Reoviridae) in the paediatric population, where dominance of one particular genotype was shown to dramatically change from year to year, at least in Central and Eastern Europe [179].

c. The question of inflammation in PCa leads to the search of a causing agent in both affected patients and experimental systems. This chapter has dwelled trough different aspects of this essential question. The presented appraisal of potential responsible agents clearly indicates today a lack of credible candidates among bacterial infections. Even for viral candidates, the previous discussion showed that Herpes Viruses (CMV, EBV, and HHV-8), Polyoma Viruses and Papilloma Viruses lack some of the essential features as triggering agents [157] [156]. For all these viruses, extensive detection studies were performed for years without reaching any consensus nor obtaining evidence for their presence but in a limited percentage of cases [157] [156]. Same negative result was finally obtained for a Retrovirus, XMRV, after much controversy and discussion [4] [45] [44] [48] [49] [180], while a previously retroviral candidate (HTLV-II) had been previously falsified in the case of CFS [181] [1]. It must be stressed that the essential feature discussed here is inflammation and the two most likely candidates in the previous list, i.e. Retroviruses and Papillomaviruses do not appear to induce inflammation as expected from analysis of PCa: I) Retroviruses are known to be capable of replicating inside cells, even without causing cytopathic effects or transformation for that matter [182]; II) for HPVs, the quoted work by Woodworth has clarified this point. He wrote [158]: "A hallmark of HPV infection is absence of an inflammatory response. Basal cells express low levels of HPV early proteins, they don't undergo lysis, and they are not rapidly recognized and destroyed by resident leukocytes such as NK cells and tissue macrophages HPV infections can persist and remain latent for long periods and may induce tolerance to HPV antigens.."

To the contrary and as described in Section II, MFV/MFRVs are strongly apoptogenic and capable of inducing strong/very strong inflammatory responses in several experimental systems [53] [1]. In preliminary experiments, we have established primary cultures from ap proximately 20 cases of PCa. In the majority of cases, cultured cells displayed extensive cytopathic effects and did not survive for extensive passages, with three exceptions. While these results confirm previous descriptions by Frank, McNeal and by De Marzo's group [125] [126] [127] [128], they also suggest that whatever factor elicited strong inflammatory mechanisms in the prostate, with cycles of hyperplasia and of necrosis, the same factor may increase its effects during in vitro culturing [55]. We are presently testing this set of PCa tumors and relative cultures (different passages) for presence of MFV/FMRVs.

d. Although never specifically tested, sialycilic acid and similar salts have been shown to be effective for the containment/ replication-inhibition of this family of viruses (reoviri-dae) [183] [184]. Any strategy or molecule capable of reducing their inflammatory responses would probably elicit similar results.

4.4. Sternness in PCa: MFV as a "Stem Cell Virus"

4.4.1. Prostate Cancer stem Cell or Cells ?

Essential aspects of PCa have been here discussed with emphasis on viral models [55] [1]. In this last section of the chapter and also in dealing with peculiar aspects of PCa carcinogene-sis in connection with an infecting virus (with MFV/MFRVs as potential candidates), the issue of Cancer Stem Cells (CSC) or stemness will be discussed. The concept of Cancer Stem Cell dates back to much ground work in the past two centuries, with several pioneers such as Julius Cohnheim and Rudolf Virkow already in the 19th century: they predicted the existence of "embryonic rests" at the origin of tumor formation [185] [186]. At the beginning of XXth century, Pappenheim hypothesized the existence of embryonic stem cells, but it was only in the second half of '900 that experimental evidence was provided for them [187]. In Toronto, in the '60s and '70s, the research of Ernest McCulloch and James Till demonstrated that only a minute fraction of myeloma cells grew in in vitro assays in order to form colonies in semisolid media [188, 189]. The Toronto school settled the basis for further work by John Dick (see later). In the same years, similar work was carried on by Robert Bruce, showing that only 1-4% of lymphoma cells did transplant into recipients [190], and by Jim Griffin, who demonstrated low clonogenic potential for Acute Myelogenous Leukaemia cells growing in methylcellulose [191]. Three additional lines of research paved the way for the final development of CSC hypothesis. 1. Mutations or translocations were discovered in cells at birth, which became markers of leukaemia-precursor cells (i.e., TEL-AML1, MLL-AF4, AML-ETO, OTT-MAL): these cells behave as leukaemia stem cells, since they could differentiate into several lineages/compartments, while additional mutations were required for ach-ievment of full-leukemogenesis [192] [193] [194] [52], 2. the work of Peter Fialkow clearly indicated clonal expansions of leukaemia stem cells in specific diseases such as CML, AML and Myelodisplastic Syndromes (MDS) [195] [196]. Most of this work was carried on using genetic markers such as G6PD, present on the X chromosomes: in females, one of the X is silenced by the so-called lyonization phenomenon (from Mary Lyon's work) [197], thus allowing to distinguish the expansion of individual clones in cases of heterozygosity (for ex., A/B alleles for G6PD) [198] [199]; 3. The work of A. Hamburger and S. Salmon in Tucson,

AZ, who also showed low frequency (1/10-3 to 1/10-4) of colony formation from solid tumours [200, 201]. These experiments were, however only partly convincing or reproducible (for example, in S. Salmon's work) and further ethical questions and concern were raised by experiments of C. Sautham and A. Brunschwig who injected harvested cancer cells into the same cancer patients, again discovering that only large numbers (i.e., 106) were capable of tumor iniriation [42]. Only at the end of the 80's and with the advent of authomated highspeed Flurescence Activated Cell Sorting (FACS) [202], the group of John Dick in Toronto was capable of convincingly and reproducibly demonstrating the existence of Leukemic Stem Cells (LSCs). This was accomplished by xenotransplantation assays, in which LSCs from AML were transplanted into Severe Combined Immunodeficient (SCID) mice, often crossed with Non-Obese Diabetic (NOD) mice, in which also the natural immune response (NK cells) is defective [203] [204]. In order to demonstrate stemness, these experiments had to prove the three essential features of stem cells, i.e. a. their capability of remaining dormant, b. their pluripotency, being capable of reproducing the full spectrum of cancer (i.e. leukaemia) phenotype; c. their capability of self-renewal by asymmetric division, thus, giving rise to both bulk tumour cells and their immature precursors [205] [206]. The paper by Bonnet and Dick in 1997 is considered the first clear-cut demonstration of the LSC concept by xenotransplantation [207]. Subsequently, the same concept (Cancer Stem Cell or CSC) was also proven in solid tumours, initially in breast cancer by Al-Hajj et al. in 2003, where the CSC was shown to be CD44+CD24-/low lineage [208]. However, additional markers were subsequently identified in breast cancer, one of the most interesting ones being Aldehyde Dehydrogenase (ALDH), which appears to affect the phenotype of cancer cells, being associated to capacity of detoxification and a more aggressive behaviour also in other types of CSC [209] [210]. ALDH however doesn't seem to be an universal marker, as it is not, for example, associated with a more aggressive phenotype in melanoma cells [211]. Another controversial issue in recent years has concerned the frequency of Cancer Stem Cells (CSC) in different tumours. For example, a recent paper by Quintana et al. calculated that with an assay employing NOD/SCID IL2Rg mice, up to 25% of melanoma cells were tumorigenic

[212]. Similar controversies are also present for the identification of prostate CSC [176] [177] [178]. In fact, two different populations of SC and prospective CSC were isolated in PCa

[213] [214]. An initial paper in Nature described regeneration of the whole prostate from a single basal cell, which in addition to classical markers of prostate cell differentiation (Sca-1+, CD133+, CD44+) also displayed presence of c-KIT receptor (CD117+) [215]. However, a subsequent paper by the group of Michael Shen convincingly showed that among lumi-nal cells, rare precursors exist which display presence of the homeobox gene NKx3-1 in absence of androgens and are therefore called castration-resistant Nkx3-1 expressing cells (CARNs) [216]. These cells can reconstitute prostate ducts after transplantation and, upon deletion of the suppressor gene PTEN, rapidly form carcinomas in vivo [216]. Finally, the group of Owen Witte has recently shown that it is also a basal cell which can initiate tumori-genesis in nude mice through cooperation of AKT, ERG and androgen receptor [217]. It is therefore possible that more than one precursor stem cell is the target of malignant transformation in prostate cancer. Furthermore, this could also fit with the described PCa carcino-genesis, in which a rather diffuse "field effect" has been known for some time [218] [219].

4.4.2. Evidence for MFV as Stem Cell Virus, possibly involved in PCa Carcinogenesis.

a. In initial preliminary experiments, we have shown that dilutions of MFV/MFRVs for several log.s (from 10-2 to 10-8 FFU/ml) will cause a similar number of transformants, thus indicating that the limiting factor was not the virus itself, but rather its target. Since an equal number of precursor stem cells are believed to be present in such cultures, it is hypothesized that the target is indeed a SC [1].

b. The Micro-Foci induced by MFV have several features of deranged stem cells, in which genetic aberrations took place, such as MYCN amplification in neuroblasts and t(8;14) / t(2;8) in paediatric lymphomas (BL-type). Even the so-called organoids or tumorspheres of PCa (prostaspheres) have similar fetures of MFs: we are now performing experiments in order to convert normal prostate tissue/cell lines into prostaspheres by MFV infection [53].

c. As mentioned, PCa is characterized by an initial oligoclonality, which underlines carci-nogenesis through a "field-effect" (FE). Evidence of oligoclonality were also obtained by molecular biology studies (see next point). However a molecular explanation for FEs is still lacking [218] [219]: MFV/MFRVs could explain FE alterations in view of the slowly progressing infection, mostly through cell-to-cell contacts [53] [55].

d. In approximately 50% of PCa, peculiar translocations TMPRSS2-ERG have been detected, which join together an androgen regulated gene: the transmembrane protease serine 2 gene, TMPRSS2, with at least 26 different genes for transcription factors [220] [221, 222]. Although data on association of translocations with PCa aggressiveness are controversial, the translocation is an excellent marker of clonality (individual breakpoints): they have shown initial existence of oligoclonal disease, further evolving into mono-clonality during metastatic disease [223].

e. We have shown in several experiments -and previously discussed in section 2- that MFV/MFRVs infection is associated or causing peculiar genetic aberrations such as MYCN amplification (I.E., Fig. 7) or t(8;14) / t(2;8) translocations in paediatric lymphoma [53]. Similarly, we hypothesize that the associated translocations induced by MFV/ MFRVs in prostate cells are TMPRSS2-ERG translocations, which would confer resistance to virus-induced apoptosis [55]. Experiments are being carried out in several PCa biopsies already characterized for presence of translocations (in 25% of cases).

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