Up to 40% of all elderly men may harbor prostate carcinomas, less than 20% develop symptomatic disease, which in about 3% becomes the cause of death. Great advances have been made in prostate cancer detection due to refined PSA-based assays, imaging and histopathology. In their wake, two questions have gained importance, i.e. which tumors represent clinically significant disease and how tumors having progressed locally or metastasized can be identified and appropriately treated. Molecular research is thus challenged to provide a reliable classification of prostate cancers and to identify targets for novel therapies in those tumors no longer containable by surgery, irradiation, and anti-androgenic therapy.

Among such aggressive prostate cancers, molecular alterations are not uniform. Instead, these tumors contain different combinations of genetic and epigenetic aberrations that each appear to influence the biological processes crucial for the cancer phenotype 123. It is therefore necessary to disentangle the influence of individual alterations by factor analysis approaches. Through defining the biological consequences of particular recurrent molecular alterations – considered as factors – it may become possible to identify subgroups of prostate carcinoma whose behavior is determined by them. These factors could then be used to predict the prognosis in each particular subgroup. In addition, elucidation of the mechanisms by which a factor exerts its effects on prostate cancer progression would provide specific targets for therapy within the respective subgroup. This approach does not imply that factors act independently of each other. Indeed, we suggest here that two molecular alterations previously identified as being associated with more aggressive prostate cancers appear to interact in a synergistic fashion during tumor progression.

The first factor, alteration of chromosome 8, is found in up to 50% of prostate cancers 123. A large number of publications have concordantly reported that chromosome 8 alterations are significantly associated with various established histopathological indicators of poor prognosis or directly with clinical outcome. Curiously, 8p losses or 8q gains both seem to have similar clinical effects (reviewed in 4). One reason for this unusual relationship is that 8q gain is often mechanistically linked to 8p loss through isochromosome 8q formation 5. Moreover, it has been difficult to trace the effect of chromosome 8 alterations to any single gene 2. Interestingly, chromosome 8 has also emerged as the site of a hereditary prostate cancer gene in genome-wide searches. A good candidate is MSR1 at 8p23.1. Inherited mutations at this locus are presumed to modify immune responses during carcinogenesis 1. Thus, the importance of chromosome 8 alterations as a factor in prostate cancer progression is undisputed, but the question of which biological processes they influence is open.

The second factor is hypomethylation of LINE-1 retrotransposons, which constitute approximately 18% of the human genome. LINE-1 hypomethylation is part of a broader process, 'genome-wide' hypomethylation that affects not only repeat sequences, but also single-copy genes inactivated by DNA methylation in normal adult tissues 6. While hypomethylation occurs at early stages in some cancer types, it is associated with tumor progression in others. Conceivably, this association is brought about by increased chromosomal instability as a consequence of repetitive sequence hypomethylation and by reactivation of specific genes favoring tumor cell survival and adaptation during invasion and metastasis 7. However, the details of the relationship between genome-wide hypomethylation and cancer progression are poorly understood. In prostate cancer, LINE-1 hypomethylation is detected in only a subset of localized cancers, but is highly prevalent in high-stage and metastatic cases 89. In contrast, hypermethylation of selected single copy genes occurs consistently at early stages of carcinogenesis 910.

The two factors, chromosome 8 alterations and LINE-1 hypomethylation, may not be independent of each other. In a previous study of 55 prostate carcinomas including many advanced stage and recurrent cases, we found LINE-1 hypomethylation to be highly significantly associated with chromosomal instability 8, in keeping with expectations from other cancer types and animal models 67. Unexpectedly, we found a particularly close relationship between hypomethylation and chromosome 8 alterations. This raised the question whether this association was (i) mechanistic, i.e. a consequence of chromosome 8 destabilization by hypomethylation or, conversely, deregulation of DNA methylation as a consequence of chromosome 8 changes, (ii) the consequence of a common factor causing both alterations, or (iii) an indication of a functional interaction between both factors that promotes prostate cancer progression. Here, we will present evidence that explanation (iii) is most likely. Our analysis revealed that the interaction of the two factors appears to relate to several biological processes already implicated in prostate cancer progression, prominently suppression of immune responses and changes in the cortical actin cytoskeleton and the extracellular matrix.

The starting point of the present investigation was our previous observation that chromosome 8 alterations and LINE-1 hypomethylation concurred in many prostate cancers 8. Such a relationship could be mechanistic in the sense that one alteration causes the other. For instance, chromosome 1 alterations may sometimes be a consequence of hypomethylation of CpG-rich satellites enriched in its juxtacentromeric heterochromatin 28. Clearly, the fact that the highly significant association was not replicated in the present study makes mechanistic relationships of this kind unlikely. The major difference in the former study was a high proportion of metastatic and recurrent cancers, which exhibited LINE-1 hypomethylation regularly and chromosome 8 alterations often. Therefore, a likely explanation for the discrepancy is that LINE-1 hypomethylation and chromosome 8 alterations both contribute to prostate cancer progression, but are brought about by independent causes.

The fact that they concur in advanced cancers suggests a synergism between the two factors that could be exerted in two ways. Each alteration could target independent biological processes contributing to tumor progression, e.g. one might promote cell proliferation, while the other blocks apoptosis. Alternatively, both factors could target the same process(es). The expression profiling experiment in the present study was designed to distinguish between these alternatives and to identify relevant biological processes. The results clearly indicate that the two alterations act upon the same processes. Very few genes were significantly different between the tumors with or without LINE-1 hypomethylation or between those with or without chromosome 8 alterations, whereas interaction analysis identified several clearly circumscribed biological processes. One prominent process can be roughly categorized as suppression of immune responses, a second one as altered cytoskeletal organization and cell adhesion. Both processes are considered generally important in carcinogenesis, especially during tumor progression. Of note, these processes were identified by the interaction analysis as particularly important in the group of cancers with both alterations. The analysis of individual genes by quantitative real-time PCR revealed that many (e.g. MX1, EPB41L3), but not all (notably BCCIP and TLR3) of the genes significant in the interaction analysis were also overexpressed generally in prostate cancers compared to benign tissues.

More specifically, most individual genes differentially expressed in the immune-response GO groups relate to the activation of innate immune responses following viral infection. A key step in this response is the production of type I interferons by infected cells. These exert antiproliferative effects themselves and initiate adaptive immune responses. Antiproliferative and proapoptotic factors from T-cells, including IFNγ, then help to eliminate infected cells. The same sequence is thought to be involved in immunosurveillance against tumors caused by viruses and other carcinogens 29. In particular, loss of responsiveness to IFNγ has previously been linked to prostate cancer progression 3031. This result is strengthened by our finding that genes associated with interferon responses are down-regulated in prostate cancer compared to normal tissue overall and even more so in a subgroup of cancers harboring molecular alterations typical of advanced stage tumors. Importantly, however, the genes identified as downregulated in our analysis include several that are involved in the earlier phase mediated by type I interferons, especially the prototypic type I interferon response genes MX1 and MX2. Of note, MX1 has a direct antiproliferative effect 32, as does GADD45α 33. IFNB1 itself and IRF3 encoding a key transcription factor for its synthesis appeared downregulated in the group of prostate cancers with both alterations, too. In addition, expression of certain toll-like receptors including TLR2 and TLR3 that recognize 'alien' structures was diminished, at least in this subgroup. Downregulation of TLR3, which detects dsRNA of viral origin and aberrantly methylated endogenous RNAs 34 is apparent from a previous prostate cancer microarray study, but not was not followed up there 15. Our findings thus suggest that the immune response to some prostate cancers may fail at at an early step because the cancers are not recognized. The finding that TLR3 down-regulation occurred in a subset of cancers and was associated with recurrence may mean that some cancers are more successful than others in escaping detection by the immune system.

Our findings are particularly intriguing when considered together with current knowledge on hereditary prostate cancer genes. The best characterized HPC1 gene encodes RNaseL, a protein involved in response to dsRNA produced during viral infections or certain other aberrant RNAs 35. Another hereditary prostate cancer gene candidate is MSR1 at 8p23 likewise involved in innate immune responses 1. Polymorphisms in TLR genes have also been implicated as predisposition factors 3637. Intriguingly, two genes (BCCIP, HMG20B) appeared in our analysis that interact with BRCA2, another, albeit weaker candidate for a hereditary prostate cancer gene 1. GADD45A found downregulated in prostate cancers in our study is a well-established BRCA1 target. BRCA1 and IFNγ together act upon the RNaseL pathway 38.

The preponderance of immune response genes among hereditary prostate cancer genes has been interpreted as indicating a role for an aberrant immune response towards a viral agent in prostate carcinogenesis 135. Obviously, our data are in line with that interpretation. Of note, the cancers displaying the most pronounced down-regulation of immune response-related genes harbored significant hypomethylation of LINE-1 retrotransposons and chromosome 8 alterations. Diminished methylation may facilitate LINE-1 reexpression 39. LINE-1 hypomethylation in cancer parallels that of HERV endogenous retroviruses 3940 and of ALU (SINE) sequences 40. Activation of endogenous retroelements occurs in response to various types of genotoxic stress and may also take place during infection by exogenous viruses (reviewed in 41). Indeed, processed HERV transcripts have been observed in prostate cancer 42. HERV proteins are recognized as autoantigens in cancer and autoimmune diseases 4143. ALU transcripts influence the dsRNA-dependent protein kinase PKR, which regulates RNaseL activation, and TLRs 44. Conceivably, therefore, what appears as an aberrant response to an exogenous viral agent in prostate carcinogenesis might reflect to some extent the activation of endogenous retroelements. Interestingly, the recent completion of the chromosome 8 sequence has highlighted an unusual concentration of genes involved in the regulation of the immune response on this particular chromosome 45. Therefore, a speculative interpretation of our findings is that increased expression of endogenous retroelements in prostate cancer cells with hypomethylated genomes would contribute to the activation of immune responses, but is tolerated as a consequence of chromosome 8 alterations. We suggest this as a working hypothesis for further studies.

Similarly, our study implies that altered expression patterns of cytoskeletal and extracellular matrix proteins in prostate carcinoma are linked to alterations of chromosome 8 and LINE-1 hypomethylation. A novel finding in this context is the involvement of 4.1 proteins in prostate cancer. These proteins contain a phosphatidyl inositol phosphate-binding domain and connect a variety of transmembrane proteins to the actin cytoskeleton, thereby organizing cell polarity and motility. The two members of the family investigated here more closely are already implicated in other cancers. EPB41L3 was proposed as a potential tumor suppressor in lung cancer 25 and was shown to be down-regulated by promoter hypermethylation and allelic loss in renal cell carcinoma 26. We report here that hypermethylation of EPB41L3 is also prevalent in prostate cancer. The 18p losses detected by CGH in 11/50 cases, could contribute to down-regulation. EHM2 encoded by EPB41L4B was identified as overexpressed in metastatic melanoma cells 46. We show here that its overexpression is common in prostate cancer. This is the first explicit study on this gene, but our findings are in line with significant differences in previous microarray data 1415. Taken together, the results suggest a shift in the pattern of 4.1 proteins associated with prostate cancer progression whose biological and clinical implications deserve further investigation.

Altered expression of Fibulin-1, which interacts with laminins reported to be downregulated in several previous studies 14151617, was previously reported only in gynaecological cancers 4748. In these cancers, FBLN1 becomes upregulated during progression. In contrast and therefore surprisingly, in prostate cancer FBLN1 appears to become generally downregulated. The decreases in laminin and Fibulin-1 expression may be related to the dissolution of the basement membrane in prostate cancer tissues. Like the 4.1 proteins, Fibulin-1 influences cell motility and polarity. As overexpression and abnormal localization in breast cancer can lead to recognition of Fibulin-1 as an autoantigen 48, its decreased expression in prostate cancers may also lower their immunogenicity.

Promoter hypermethylation underlies GADD45A downregulation in breast cancer 27. In prostate cancer altered methylation at a more distant, but unfortunately unspecified site in the gene has been reported 49. We found the actual promoter unmethylated in prostate cancer tissues and cell lines. Induction of GADD45A by SAHA treatment in prostate cancer cell lines could mean that downregulation is associated with histone deacetylation and altered chromatin structure. The additive effect of 5-aza-dC treatment might reflect the methylation at the distant site 49.

LINE-1 hypomethylation and chromosome 8 alterations are commonly associated in highly advanced prostate cancers. The present study suggests that both alterations do not cause each other. Rather, they appear to act as converging and even synergistic factors contributing to prostate cancer progression. Interaction analysis identified suppression of innate immune responses and cytoskeletal and extracellular matrix changes as common targets. This implies that candidate tumor genes on chromosome 8 as well as DNA hypomethylation should be considered for their influence on these processes in prostate cancer. Intriguingly, many hereditary prostate cancers genes are also involved in regulation of innate immune responses. The data stress the importance of altered cell adhesion and cytoskeletal organization in prostate cancer and specifically implicate changes in 4.1 protein expression in this process. The 4.1 protein encoding genes and several others newly identified here ought to be investigated for their usefulness in prostate cancer detection and classification through expression or methylation analyses.

The author(s) declare that they have no competing interests.

WAS, BW, and JR conceived the study. WAS and JR drafted the manuscript. VJ performed the CGH analysis evaluating it together with BW. CH, MJH, MY, SF, AW, and ARF carried out and evaluated the expression and methylation analyses with guidance by WAS. AA, TL, and JR performed the bioinformatic analyses. MM contributed the clinical data and according statistical evaluations. RE controlled the tissue sampling and histopathological data. All authors read and approved the final manuscript.