Multiple pathogens and prostate cancer

The aim of this review is to consider whether multiple pathogens have roles in prostate cancer. We have reviewed case control studies in which infectious pathogens in prostate cancer were compared to normal and benign prostate tissues. We also reviewed additional evidence from relevant published articles. We confirmed that high risk human papilloma viruses are a probable cause of prostate cancer. We judged Escherichia coli, Cutibacterium acnes, Neisseria gonorrhoea, Herpes simplex, Epstein Barr virus and Mycoplasmas as each having possible but unproven roles in chronic prostatic inflammation and prostate cancer. We judged Cytomegalovirus, Chlamydia trachomatis, Trichomonas vaginalis and the Polyoma viruses as possible but unlikely to have a role in prostate cancer. The most influential cause of prostate cancer appears to be infection induced chronic inflammation. Given the high prevalence of prostate cancer it is important for action to can be taken without waiting for additional conclusive evidence. These include: Encouragement of all boys (as well as girls) to have HPV vaccines The vigorous use of antibiotics to treat all bacterial pathogens identified in the urogenital tract The use of antiviral medications to control herpes infections Education about safe sexual practices Encouragement of all boys (as well as girls) to have HPV vaccines The vigorous use of antibiotics to treat all bacterial pathogens identified in the urogenital tract The use of antiviral medications to control herpes infections Education about safe sexual practices


Introduction
The aim of this review is to consider whether multiple pathogens have roles in prostate cancer. Multiple pathogens have long been hypothesised as an underlying cause of prostate cancer. However, apart from high-risk for cancer human papilloma viruses (HPVs), no specific pathogens have confirmed causal roles.
We have previously shown that high risk for cancer human papilloma viruses have a probable, but not conclusive, causal role in prostate cancer [1]. This is important because of the availability of safe and effective vaccines against HPV infections. In this review we have updated the evidence which may implicate other infectious pathogens.
We consider it is unlikely that any acute infectious pathogens cause prostate cancer. On the other hand, infectious pathogens that cause long term chronic inflammation are likely to have roles in prostate cancer.

Epidemiology
Prostate cancer develops in 1 in 8 Western men [2]. About 60% of cases occurs in men aged 65 years or older. It is rare in men under the age of 40 years. About 30% of men have undiagnosed prostate cancer at the time of their death, hence the saying "many men die with, rather than from, prostate cancer". Prostate cancer occurs more frequently in Western than Asian men [2]. When Asian Page 2 of 14 Lawson and Glenn Infectious Agents and Cancer (2022) 17:23 men migrate from low to high risk countries the risk of developing prostate cancer increases [3]. The reason is not known. However, the number of immigrants developing prostate cancer is still lower than that of men in Western countries [4]. This phenomena is also present in breast cancer for Asian women who migrate from low to high risk countries, the risk of breast cancer rapidly increases within two generations to almost equal that of the host country [5].

Methods
We have conducted a review of selected English language publications listed in PubMed from 1960 to 2021 relevant to infectious pathogens and prostate cancer. Only studies which included controls were reviewed. Any form of selection introduces bias. For this reason the two authors independently selected the studies that were considered. Any differences in the selection were discussed and joint decisions were made. Additional problems in the assessment of the role of specific pathogens in prostate cancer include (1) the variations in outcomes of studies using similar methods in the same populations, (2) contamination of the prostate specimens and (4) the absence of benign or normal prostate controls. The selection of pathogens for this review was based on the many previous studies of infections and prostate cancer. These pathogens included Human papilloma viruses, Cetabacterium acnes, Herpes viruses including Epstein Barr virus, Neisseria gonorrhoea, Herpes simplex, Epstein Barr virus, Cytomegalovirus, Chlamydia bacteria, Trichomonas bacteria, Mycoplasmas and Polyoma viruses. Case control studies were available for each of these pathogens. Other pathogens, for which no case controls have been conducted, may also have roles in prostate cancer, for example Escherichia coli, fungal prostatitis, mouse mammary tumour virus and human immunodeficiency virus [6,7].
The use of case control studies for the study of infections and prostate cancer can be misleading. This is because in most studies the non-cancer controls were benign prostate tissues. Chronic infections are common in the prostate and this can negate the comparisons between cancer and controls.
The Bradford Hill criteria have been frequently used for assessing causal roles of pathogens and other agents [8]. These criteria have been immensely influential. They have largely replaced the famous Koch postulates. Over the last 50 years, it has been estimated that over 100,000 published articles have used the Hill criteria [9]. Hill developed nine criteria in the context of his research into the links between tobacco smoking and lung cancer [10]. At that time the role of viruses in various human cancers was not known. In addition, since 1965 there have been major developments in knowledge and technology. It has also been realised that the relevance of the individual criteria vary according to the nature of the pathogen or harmful agent. Accordingly, there has been a need to add and modify the classic Hill criteria. The list of the Hill and extended criteria in some order of importance include: (1) Identification and history of the candidate pathogen. (2) Epidemiology. (3) Strength of the association between the pathogen and prostate cancer. (4). Temporality (timing) of the association which includes evidence of infection by a pathogen in normal tissues before the development of the cancer. (5). Does exposure to the pathogen lead to infection, oncogenesis and cancer? (6) Experimental evidence, for example, capacity of the pathogen to cause cancer in experimental animals, capacity to infect human cells, ability to transform normal human cells into malignant cells, evidence that a vaccine or therapy can inhibit the pathogen from infecting or transforming cells. (7) Coherence, analogy, biological plausibility. (8) Transmission including identification of the source and means of transmission of the pathogen. (9) Oncogenic mechanisms. (10) Multiple viral and causal factors. (11) Specificity-this criteria was in Hill's original list but is rarely helpful as many viruses and other pathogens can lead to cancer in different organs.
Hill [8] strongly cautioned against dogmatism. " None of my nine viewpoints can bring indisputable evidence for or against the cause-and-effect hypothesis and none can be required as a sine qua non (meaning an essential requirement).
In this current review these criteria could only be fully used with respect to human papilloma viruses because of the limited evidence available for the other pathogens listed above.

Human papilloma viruses (HPV)
We have recently reviewed the evidence and concluded that it is highly likely that high risk for cancer HPVs have a causal role in prostate cancer [1]. The most important evidence is the demonstration that the prevalence of high-risk HPVs is consistently higher in prostate cancer than in benign prostate controls. This is shown in Table 1  . In brief the evidence is as follows: 1. High risk for cancer HPVs have been identified in many countries by a range of methods in normal, benign and malignant prostate tissues [37]. 2. In 10 of 27 case control studies conducted with PCR techniques, the prevalence of high-risk HPV DNA was significantly higher in prostate cancers as compared to normal and benign prostate controls (studies in which HPVs were not identified have not been included in Table 1). In these 27 studies there were 399 HPV positive of 1678 prostate cancers (24%) and129 HPV positive of 1331 benign prostate controls (10%) (p = 0.001). 3. High risk HPV types 16 and 18 have the capacity to immortalise and transform normal prostate cells into malignant cells [38,39]. 4. HPVs are mainly transmitted by sexual activity [40].
HPVs can be transmitted throughout the body via circulating extra-cellular vesicles and blood [41]. 5. High risk HPVs are associated with inflammatory prostatitis which can lead to benign prostate hyperplasia and later prostate cancer [42,43]. 6. High risk HPVs of the same type have been identified in benign prostate tissues 1-11 years before the development of HPV positive prostate cancer in the same patients [44].
While the highest prevalence of HPV genital infections occurs in younger people there is an increased prevalence in older age groups (over 55 years) [45,46]. This increase in older people is unlikely to be due to increased sexual activity. Prostate cancer is much more prevalent in older men. Accordingly there may be an association between older age HPV reactivation and prostate cancer.
The reason for the reactivation of HPVs is not known. An explanation may be the concept of "trained immunity" [47]. This concept involves the long-term reprogramming of innate immune cells, which can be reactivated by stimuli such as infections or chemicals. While this response can be protective against a harmful stimulus, over-reactions such as inflammation can develop. In turn, chronic inflammation can be oncogenic. While there is no direct evidence available with respect to prostate cancer, HPVs can remain dormant in the host cell genome, thus evading the host immune response until they are reactivated [48]. The oncogenic mechanisms for HPV oncogenesis in prostate cancer are not clear and may differ from HPV oncogenesis in cervical cancer. There is evidence that HPV E7 oncogenic proteins may be directly involved early in prostate oncogenesis [17]. HPV infections may have an indirect role by inhibiting the protective function of APOBEC3B enzymes against other virus infections [49,50].
Effective and safe vaccines are available for the prevention of a wide range of different types of HPV infections [51].
With respect to Silvestre et al. [22], Tachezy et al. [26] and Mokhtari et al. [28] the numbers of positive cases are too few to justify statistical analysis.

Cutibacterium (Propionbacterium) acnes
Cutibacterium acnes (C. acnes) are part of the commensal flora of the skin where they colonize hair follicles and sebaceous glands [52]. Different types of C. acnes can also cause serious post-operative infections. Cutibacterium acnes may also be present in the urogenital tract including the prostate. Cutibacterium acnes can damage blood cells, cause host tissue degradation and disrupt cell surface components.
Cutibacterium acnes has been identified in prostate cancer tissues. In 2 of 6 case control studies C. acnes was significantly more prevalent in prostate cancer than in control benign prostate tissues (Table 2) [53][54][55][56][57][58]. Most C. acnes from prostate cancer tissues differ genetically from common skin C. acnes [59]. Alexeyev et al. [53] have identified C. acnes in benign prostate tissues taken up to 6 years apart from individual subjects. This indicates that C. acnes infection can be chronic and a cause of chronic inflammation. Cutibacterium acnes infections induce upregulation of inflammatory genes and cytokine secretion in prostate epithelial cells [60].
Accordingly C. acnes is a candidate pathogen in prostatitis and prostate cancer.
The evidence that antibiotics can control C. acnes infections is based on skin infections [61]. Resistance to antibiotics is an increasing problem.

Escherichia coli
Escherichia coli have been consistently identified by PCR and Next Generation Sequencing in prostate cancer and benign prostate tissues [54,62]. Unfortunately, good controls have not been used in these studies and no case control studies have been identified. A problem in studying E. coli and prostate cancer is that biopsies are usually conducted by gaining access to the prostate via the rectum. This can cause contamination of the prostate tissues by rectal located E. coli.
Escherichia coli is usually a harmless commensal bacteria that colonizes the human gut. However, many different types and strains exist, some of them have virulence properties that can result in inflammation and damage of the prostate. Jain et al. [63] have isolated E. coli from benign prostate tissues and demonstrated that this pathogen activated NF-kB and induced damage to normal cultured prostate epithelial cells. NF-kB proteins are activated by carcinogens and are known to be involved in oncogenesis [64]. Hemolysin and necrotizing factor type 1 occur significantly more frequently among C. coli isolates causing prostatitis than among those causing cystitis or pyelonephritis [65].
It is considered likely that some types of E. coli have causal roles in colon cancer [66]. Accordingly it is possible that E. coli can also cause prostate cancer.

Neisseria gonorrhoea (N. gonorrhoea)
Neisseria gonorrhoeae is the well known cause of the sexually transmitted disease gonorrhea [67]. The organism can manipulate the immune response which leads to a lack of protective immunity. Therefore individuals can become repeatedly infected. Gonorrhoea is generally a mucosal infection of the urethra with a pustular discharge. More severe sequalae include salpingitis and pelvic inflammatory disease which may lead to sterility and/ or ectopic pregnancy. Neisseria gonorrhoeae can cause chronic inflammation of the prostate which in turn can be oncogenic [68]. Gonorrhoea is susceptible to an array of antibiotics. Antibiotic resistance is becoming a major problem.
There have been 22 case control studies in which the prevalence of N. gonorrhoea in prostate cancer has been compared to controls (Table 3) . In six of these studies it was shown that N. gonorrhoea was significantly more prevalent in the prostate cancer cases. In 16 of these studies there was no significant difference been the cases and controls.
There is a possible explanation for these conflicting data, namely that sexually transmitted diseases are frequently due to multiple pathogens. In the meta-analysis by Taylor et al. [91] there were significant correlations between both N. gonorrhoea and HPVs and increased prevalence of prostate cancer (odds ratios gonorrhoea 1.35, HPV 1.39). It is possible that high risk HPVs were the cause of prostate cancer in these studies and that N. gonorrhoea was also present but not oncogenic.
HSV-2 is associated with anogenital infections and is a sexually transmitted infection.
Both virus types can cause both kinds of infection. Infections due to herpes simplex do not usually confer immunity. No vaccines are currently available.
In four of 12 studies Herpes simplex 1 or 2 were significantly more prevalent in the prostate cancer cases (Table 4) [70,87,88,[92][93][94][95][96][97][98]. Dennis et al. demonstrated that herpes simplex 2 could be identified in prostate cancer tissues over a period of 8 years [98]. These findings suggest that if herpes simplex has an oncogenic capacity there may be a long latency period for prostate cancer development after HSV-2 infection.
Acyclovir has been successfully used to treat genital herpes simplex infections [99].
There have been four case control studies of EBV and prostate cancer. In one study by Sfanos et al. [54], EBV was significantly more prevalent in prostate cancer compared to controls (Table 5) [54,97,100,101].
The effectiveness of antiviral agents (acyclovir, valomaciclovir and valacyclovir) in acute infectious mononucleosis is uncertain [99,102].

Cytomegalovirus (CMV) (herpes virus 5)
Human CMV is present in over 80% of most populations. Transmission can occur during foetal life, via breast milk, saliva and during sexual activities. Human CMV infections in healthy people are mostly mild or without symptoms. In contrast, CMV can cause serious defects during foetal life and life threatening illness among immunocompromised patients such as transplant recipients and patients with AIDS [103].
As shown in Table 6 [23,87,93,104,105] in four of five case control studies there were no significant differences between the prevalence of CMV in prostate cancers and controls. In one study CMV was identified in the controls but not in prostate cancers [23].

Chlamydia trachomatis (C. trachomatis)
Chlamydia trachomatis is a common, sexually transmitted bacteria. Chlamydia trachomatis initiates and can maintain inflammation and persistent infection including prostatitis [105]. Human prostate cancer epithelial cells are susceptible to C. trachomatis infection and initiate inflammation [106,107]. As inflammation is associated with prostate cancer it has been hypothesized that C. trachomatis could have a causal role.
However, as shown in Table 7 [81,87,88,98,106,[108][109][110] in eight case control studies there were no positive associations between C. trachomatis infections and prostate cancer. On the other hand, all these studies are based on serology, and it is possible that these case control studies are misleading as C. trachomatis may be causing chronic infection in the prostate leading to prostate cancer. This would lead to positive antibodies in both benign prostate controls and prostate cancer.  Azithromycin and Doxycycline antibiotics appear to be effective in the treatment of sexually transmitted C. trachomatis [111].

Trichomonas vaginalis (T.vaginalis)
Trichomonas vaginalis is a common protozoan infection frequently transmitted during sexual activities [112]. Trichomonas vaginalis in men is usually asymptomatic but may cause urethritis, prostatitis, epididymitis and infertility [113].
As shown in Table 8 [86,[114][115][116][117][118][119][120][121] in eight of nine case control studies there is no increase in risk of prostate cancer in association with T. vaginalis infections. In two studies positive antibodies were higher in the controls than the cancer. These nine studies were all based on serology and involved a high number of subjects.
In a large serology based study by Tsang et al. [122] there was no increase in prostate cancer deaths associated with T. vaginalis. This finding makes it unlikely that T. vaginalis is associated with prostate cancer.
The 5-nitroimidazoles (metronidazole, tinidazole, secnidazole) are the only class of antimicrobials effective against T. vaginalis [113]. Unfortunately, there is growing concern over drug resistance with metronidazole.
Of particular interest are the studies based on PCR analyses of tissues as compared to studies based on serology. Three of the PCR studies with positive results were significant, and two showed a trend that Mycoplasma infections were more frequent in prostate cancers than benign prostate controls. Accordingly, it is possible that Mycoplasma bacteria may have a role in prostate cancer. However additional evidence is required.
Antibiotics can be effective in treating Mycoplasma bacterial infections. Unfortunately, resistance to antibiotic treatment is emerging [130].

Polyoma viruses (hPy)
The two human polyomaviruses (hPy), BK virus (BKV), and JC virus (JCV), are commonly present in human populations. Infections usually occurs in childhood but rarely cause clinical symptoms. In immunocompromised patients JCV can cause serious neurodegenerative conditions. There is no direct evidence that hPy viruses are oncogenic [131].
In two small studies based on PCR there was a significant association with prostate cancer. There were no significant associations in 9 studies.
Accordingly it is unlikely that these polyomaviruses have causal roles in prostate cancer.

Fungal prostatitis
Infections of the prostate by several fungi are the unusual cause of prostatitis. These fungi include Blastomycosis, Candida albicans and Cryptococcus [140]. There is no evidence that these fungi are associated with prostate cancer. However, there must be suspicions about any pathogen which leads to chronic inflammation.

Mouse mammary tumour virus (MMTV)
MMTV is the proven cause of breast cancer in mice. There is compelling evidence that MMTV-like viruses are also causal in human breast cancer [7]. MMTV has been identified in prostate glands of mice [141]. MMTV-like viruses have been identified in human prostate cancers [6]. However, no studies have been  conducted to determine if MMTV is causal in human prostate cancer.

Human immunodeficiency virus (HIV)
Compared to the general population, people living with HIV have a lower prevalence of prostate cancer [142,143]. This is probably due to the suppression of immune related B and T cells associated with both HIV and MMTV infections.

The gut microbiome and prostate cancer
The gut microbiome may also play an indirect role in various cancers [144]. In a study which compared the gut microbiota in men with prostate cancer and benign controls there was a significant difference in gut microbiol composition [145]. The meaning of these observations is not known.

Discussion
High risk human papilloma viruses are the only pathogens for which there is sufficient evidence to indicate a probable causal role in prostate cancer. Fortunately, there are safe and effective vaccines available to prevent HPV infections [146].
Other pathogens may have roles in prostate cancer but the evidence is limited. These include Cutibacterium acnes, Neisseria gonorrhoea, Herpes simplex, Epstein Barr virus, and Mycoplasmas. In our view it is unlikely that Cytomegalovirus, Trichomonis vaginalis, Chlamydia trachomonis, Polyoma viruses, Human immunodeficiency virus and fungi have causal roles in prostate cancer.
HPVs are the only pathogen considered in this review which have a proven oncogenic capacity. However, in its acute stage it is unlikely that an HPV infection leads to prostate cancer as HPV infections are common in young men and prostate cancer occurs mainly in older men. On the other hand, as considered above, the influence of HPV may be reactivated and lead to prostate oncogenesis via long-term reprogramming of innate immune cells.
While the oncogenic mechanisms probably differ between these pathogens, of particular relevance is the potential role of inflammation in prostate cancer. Different pathogens may each cause chronic inflammation. Multiple pathogens are frequently present in prostate tissues and chronic exposure can lead to chronic inflammation and ultimately to prostate cancer. The relevant evidence has been reviewed in detail by De Bono et al. [147] and Gobel et al. [148].
A precise mechanism linking inflammation to cancer is the nuclear transcription factor "kappa-light-chainenhancer" of B-cells known as NF-kB. This is a protein activated by many carcinogens. It controls genes commonly associated with oncogenesis [64]. Almost all infectious agents linked with cancer activate NF-nB. This has been confirmed experimentally in mice by the inactivation of NF-kB which reduced inflammation initiated cancer formation [149]. Infectious pathogens can activate inflammatory pathways which lead to genomic instability in tissue cells which in turn lead to malignant transformation. HPV, human herpes virus, and EBV, have been specifically shown to activate NF-kB. Confirmation of this evidence has been provided by the reduction in risk of cancer by anti-inflammatory agents such as aspirin [150].

Conclusions and actions
The most influential cause of prostate cancer appears to be infection induced chronic inflammation. Given the high prevalence of prostate cancer it is important for action to can be taken without waiting for additional conclusive evidence. These include: