The Relationship between Mycoplasmas and Cancer: Is It Fact or Fiction ? Narrative Review and Update on the Situation

More than one million new cancer cases occur worldwide every year. Although many clinical trials are applied and recent diagnostic tools are employed, curing cancer disease is still a great challenge for mankind. Heredity and epigenetics are the main risk factors often related to cancer. Although, the infectious etiological role in carcinogenesis was also theorized. By establishing chronic infection and inflammation in their hosts, several microorganisms were suggested to cause cell transformation. Of these suspicious microorganisms, mycoplasmas were well regarded because of their intimate parasitism with host cells, as well as their silent and insidious role during infections. This assumption has opened many questions about the real role played by mycoplasmas in oncogenesis. Herein, we presented a sum up of many studies among the hundreds which had addressed the Mycoplasma-cancer topic over the past 50 years. Research studies in this field have first started by approving the mycoplasmas malignancy potential. Indeed, using animal models and in vitro experiments in various cell lines from human and other mammalians, many mycoplasmas were proven to cause varied modifications leading to cell transformation. Moreover, many studies have looked upon the Mycoplasma-cancer subject from an epidemiological point of view. Diverse techniques were used to assess the mycoplasmas prevalence in patients with cancer from different countries. Not less than 10 Mycoplasma species were detected in the context of at least 15 cancer types affecting the brain, the breast, the lymphatic system, and different organs in the genitourinary, respiratory, gastrointestinal, and urinary tracts. Based on these revelations, one should concede that detection of mycoplasmas often linked to ‘‘wolf in sheep's clothing” is not a coincidence and might have a role in cancer. Thorough investigations are needed to better elucidate this role. This would have a substantial impact on the improvement of cancer diagnosis and its prevention.


Introduction
Cancer is a devastating disease presenting an immense burden to humanity. According to latest statistics of the International Agency for Research on Cancer affiliated to the World Health Organization, barely 18 million new cancer cases were globally recorded in 2018, of which approximately 10 million have led to death [1]. Many causes and risk factors have been pointed to promote carcinogenesis establishment and development. Among these factors, some infectious agents have been suspected. In fact, according to the American Cancer Society, up to 20% of cancers worldwide have been related to infectious agents. e causal relationship between different types of cancer and many oncoviruses such as papillomavirus, hepatitis B virus, and Epstein-Barr virus and bacteria such as Streptococcus bovis, Salmonella typhi, Chlamydia pneumoniae, Bartonella, and Helicobacter pylori has previously been well documented [2][3][4][5][6][7][8][9][10][11][12]. One of the suspected prokaryotes in malignancy are mycoplasmas. As these atypical bacteria are notorious for their capacity to implement a low-grade chronic inflammatory condition during cell infection without compromising viability, they were thought to be ideal for promoting cancer transformation [13]. Actually, the oncogenic potential and role of mycoplasmas in cancer development have been started to be investigated since the 1950s. Mycoplasmas were first detected in leukemia patients, and later, many studies have reported their identification in variant solid cancers either directly by PCR amplification of specific DNA segments or indirectly by evaluation of their antibody status in patients. roughout this review, we will attempt to highlight the Mycoplasma-cancer relationship. Twenty years from the first review describing this relationship [14] and ten years after the second [15], we are today recalling what has been performed in this regard along with the new findings.

Strategy Research
We have conducted this review by synthesizing the results of research studies that consider Mycoplasma as an infectious agent involved in cancer. is was done by summarizing the available data with regard to the impact of mycoplasmas on the morphology, properties, and signaling pathways in mammalian cells and listing the different cases of cancer in which Mycoplasma species have been identified.
is review has been elaborated on the basis of screening in PubMed/MEDLINE databases, all types of published articles (research articles, reviews, and case reports) which have approached the Mycoplasma-cancer topic. is synthetic study was further complemented and refined by scanning the reference lists of the selected articles. All the bibliographical sources included in this review were carefully consulted and approved by all the authors.
In addition to the introduction and the conclusion, the main body of this review was conceived in three sections. e first is devoted to a general and concise description of distinctive features of mycoplasmas that made them suspicious infectious agents in cancer. To prove their malignancy potential, we have drawn on 27 studies, the synthesis of which constituted the content of the second section. e third section includes the first seven studies published between 1965 and 1970, which reported the presence of mycoplasmas in cancer patients, and 27 more recent studies published between 1995 and 2020.
ese have provided epidemiological information on the identified mycoplasmas in many types of cancer in several countries.

Mycoplasmas: Atypical Tiny Microorganisms Capable of Causing Serious Troubles
Mycoplasmas are atypical bacteria widespread in nature as parasites of human, mammals, reptiles, fish, birds, arthropods, and plants [16]. Mycoplasmas were first thought to be viruses given their reduced genome, minute size, and the total lack of a cell wall around their cytoplasmic membrane. ey are considered as the smallest self-replicating prokaryotes [17]. e extensive genome reduction, through a process of reductive evolution, has limited the mycoplasmas biosynthetic capacity and hampered their metabolic pathways [18,19]. As a result, mycoplasmas are rendered completely dependent on their hosts to acquire essential precursors such as nucleotides and amino acids to insure their survival [20][21][22][23]. Mycoplasmas have tropism for many types of eukaryotic cells, and most of them are extracellular. However, host cell invasion was reported for some Mycoplasma species such as Mycoplasma fermentans and Mycoplasma penetrans [24,25].
e investigation of this oncogenic potential has been started from the middle of the 1960s, when several studies reported the detection of some human Mycoplasma species or their antibodies in patients suffering from leukemia [51][52][53][54][55][56][57]. Although these studies did not provide causal evidence for the involvement of mycoplasmas in cancer, they represented an opportunity for further investigations.

Malignancy Potential of Mycoplasmas: Evidence In Vitro and In Vivo
After the first reports suggesting the existence of a relationship between mycoplasmas and cancer, many experiments have subsequently been carried out to determine whether these bacteria are really endowed with oncogenetic properties or they are fortuitly identified in patients diagnosed with cancer. Other studies have attempted to explore whether mycoplasmas are directly involved in the onset of cancer or rather in its progression. Many cellular and molecular mechanisms of malignancy transformation were proposed for mycoplasmas. For example, Paton et al. have shown that following serial passages of human diploid WJ-38 stem cells from normal embryonic lung and their infection with Mycoplasma orale, the growth of these cells regresses with noticeable chromosomal aberrations.
is is the first study reporting such chromosomal disorders associated with Mycoplasma infection in normal human cells [58]. Malignant transformation induced by mycoplasmas was also reported to occur in blood cells (PBMCs) [59] and in many other human cell lines from different organs such as leiomyosarcoma cell line SK-UT-1B in the uterus [60], adenocarcinoma A549 cells in the lung [61], BPH-1 cells in the prostate [62], and neuronal cell line BE-M17 [63]. Besides human cells, studies have also demonstrated that some Mycoplasma species are effective in mediating changes in other mammalian cells in mouse, monkey, and hamster. e time of inoculation and the passage level were shown to affect transformation of infected mammalian cells [64,65]. ese findings were consolidated by another similar study fulfilled by Tsai et al., by exploring the impact of infection with M. fermentans and M. penetrans species on mouse embryo cells C3H. In this study, a Mycoplasma-mediated oncogenesis was proved to take place in progressive steps and not in an acute way. In reality, the phenotypic changes noticed become progressively more conspicuous with the persistence of the Mycoplasma infection [13]. ese two Mycoplasma species were also shown, in another study, to infect the murine 32D hematopoietic cell line, which is known to undergo interleukin-3-dependent apoptosis. It was found that mycoplasmas infection altered the properties of the 32D cells, preventing their apoptotic capacity, and enabling them to grow autonomously, independently of interleukin-3 stimulation. is was evidenced by the formation of tumors after injection of transformed 32D cells into nude mice [50]. It has also been found that the properties of stem cells are affected and altered by Mycoplasma infection [66].
Some researchers have been rather interested in studying the impact of activation or inactivation of Mycoplasma infections on the expression of target genes in different cells.
ese targets included oncogenes, tumor suppressors, proinflammatory cytokines, and growth factors. Zhang et al. have reported that chronic infection with the two human Mycoplasma species M. fermentans and M. penetrans is associated with an overexpression of H-ras and c-myc oncogenes in C3H embryo cells, which involved malignant cell transformation in mouse [67]. Subsequently, other scientific teams from the same laboratory have examined and compared the expression profiles of many genes (38 cytokine genes and 1185 other genes involved in oncogenesis, apoptosis, cell growth, and cell cycle regulation) in different human and mouse cells, before and after infection with different urogenital mycoplasmas. ey concluded that distinctive mycoplasmas may have different effects (sometimes opposite) on the expression of a given gene. ese effects might progressively but significantly alter several biological properties of mammalian cells (obvious changes in cell morphology or growth rate) and thus lead to malignant transformation [68,69]. Using different approaches, Liu and Shou have also demonstrated that Mycoplasma infection has several effects on mammalian cells. ese include genome breakdown and dysregulation of the expression of certain genes involved in apoptosis and tumorigenesis [70]. Furthermore, the repercussion of mycoplasmas infection on major regulation mechanisms of programmed cell death was also investigated. Logunov et al. have monitored the effects of M. fermentans, M. arginini, M. hominis, and M. arthritidis on the p53 tumor suppressor and the nuclear factor kappaB (NF-κB) pathways, both involved in the maintenance of the cell cycle stability. Experiments carried out in vitro on a panel of human and mice cell lines showed that Mycoplasma infection inhibited p53 activity and activated the NF-κB, which are specific traits of human tumor cells. Moreover, it has been found that the same Mycoplasma species mentioned above cooperates with some oncogenes in cell transformation. However, the involved Mycoplasma protein has not been identified [71]. is was not the case in other studies identifying a M. hyorhinis-specific protein, p37, which promotes the invasiveness of the pathogen into the human cells.
is demonstrates its involvement in the malignancy process [72][73][74]. A significant association between this Mycoplasma species and tumorigenesis was once again proved in vitro and in vivo in another study [75]. is was further strengthened by two more recent research studies supporting the role of M. hyorhinis in malignant transformation of gastric cells. is is achieved either by the activation of the NLRP3 inflammasome, which mediates the maturation of proinflammatory cytokines [76] or by activating the β-catenin signaling pathway, which is crucial for tissue homeostasis [77]. Furthermore, another way for the implication of mycoplasmas in cancer promotion was proposed. Mycoplasmas were shown to disturb the anticancer process either by causing resistance to the anticancer drugs or by repression of the natural killer cells activity. In two studies realized by two teams from the University of Maryland in the USA, the Mycoplasma chaperone DnaK was shown to have oncogenetic properties that render it responsible for the potency decrease of some proteins associated with critical cellular pathways, leading to a decline in the efficiency of anticancer drugs [78,79]. Likewise, a group of Russian and German scientists have reported that sensitivity of tumorous cells to anticancer decreases after Mycoplasma infection. e authors have precisely worked with M. hyorhinis species [80]. In a Singaporean-Taiwanese study that came out just since few months, Mycoplasma infection was used to give rise to an artificial chronic inflammation condition in order to understand the influence of macrophages on natural killer cells in a context of a cancer. Results have shown that cancerous tissues infected by mycoplasmas were protected by macrophages from the attack of natural killer. Mycoplasmas have served as an example to prove that infection-induced inflammation helps in the cancer progression through natural killer cell repression moderated by macrophages [81].
As reported above, both in vitro and in vivo experimental data provided evidence that some mycoplasmas are able to affect the accuracy of genomic transmission during cell division and to disturb the coordination between cell cycle checkpoints. Many cases of chromosomal abnormalities (loss and translocations), karyotypic changes, and morphological modifications have been associated to Mycoplasma infections. More details about the above selected experimental studies are given in Table 1. As well, a schema assembling the different effects caused by Mycoplasma infections on cell lines leading to malignant transformation is presented in Figure 1.  Journal of Oncology Journal of Oncology 5 investigations based on molecular and serological detection of these bacteria in different types of cancers have been documented in many countries around the world ( Table 2).

Prostate Cancer.
Prostate cancer is the second most commonly diagnosed cancer and the sixth cause of cancer death in men worldwide. Incrimination of urogenital mycoplasmas in this type of cancer has been evoked since the mid-twentieth century [110,111]. Based on this suggestion, some studies have been conducted later to experimentally prove the mycoplasmas potential in malignant transformation of prostate cells [62,73]. More recently (during the last 2010-2020 decade), this hypothesis was further sustained by several studies reporting the detection of mycoplasmas (by PCR or RT-PCR) or their antibodies (using ELISA technique) in patients suffering from prostate cancer. Different Mycoplasma species, notably M. hominis, M. genitalium, and Ureaplasma urealyticum, were identified with variable percentages in patients from different nationalities such as Russian, Turkish, Australian, Iranian, and Japanese [82][83][84][85][86][87]. In the same context, our team has recently shown the presence of U. parvum and U. urealyticum species in a cohort of Saudi patients diagnosed with prostate cancer with rates of 20% and 16%, respectively [88]. It is important to notice that although the epidemiological data reported in these studies are interesting and persuasive, a luck of statistical and/or experimental evidences in some of them constitutes a limiting factor in the approving of the etiopathological role of mycoplasmas in prostate cancer.

Gastric Cancer. Malignant transformation in stomach
cells is also one of the most frequently occurring cancers worldwide. In addition to the risk factors often mentioned in stomach cancer, such as smoking and lack of healthy nutrition, Helicobacter pylori infection was well established as a   Journal of Oncology 7 cause of this cancer. Indeed, it is the most and best studied case illustrating the direct involvement of an infectious agent in cancer [10][11][12]. is has encouraged further investigations to determine the potential involvement of other infectious agents in this type of cancer such as mycoplasmas. Results of studies aiming to seek for Mycoplasma species in patients with gastric cancer have shown the frequent detection of M. hyorhinis.
is Mycoplasma species was identified by the Southern blot technique using specific Mycoplasma rDNA probes in 11 out 23 Japanese patients with gastric cancer (48%) [89]. Higher detection rates around 56% [90] and 54.1% [91] were reported in tumors from Chinese patients using the immunohistochemistry assay. A higher rate of 63.9% was obtained with nested PCR [92]. Interestingly, this last study also reported the detection of another Mycoplasma species, M. fermentans, however, always in conjunction with M. hyorhinis in the recruited patients. e authors of this study even succeeded in culturing these species on some cancerous specimens.
is constitutes an irrefutable evidence of their existence in cancerous tissues.

Ovarian Cancer.
According to the Global Cancer Observatory, nearly 300 thousand new cases of ovarian cancer were recorded in 2018. With about 185 thousand deaths, this type of cancer is considered one of the most deadly of the gynecological cancers [1]. Several risk factors contribute to the development of this type of cancer, the genetic predisposition being the most suspected [107,112]. However, the involvement of some urogenital microorganisms, such as Chlamydia trachomatis, in the etiology of ovarian cancer has also been raised. Such a suggestion has been investigated on the basis of the frequent detection of Chlamydia trachomatis, which is marked by a persistent infection in the female upper genital tract [113,114]. Endowed with the same properties, mycoplasmas were also suspected and their association with ovarian cancer was assessed in some studies. Indeed, in one of the investigations carried out, a combined PCR-ELISA method was designed for the detection of 15 Mycoplasma species in women with ovarian cancer. Mycoplasma infection was identified in more than half of 27 patients tested, which is a significant rate of about 60% [93]. Using the same combined procedure, Kim et al. found a rate of only 13.8% (4 among 29 Korean patients with ovarian cancer) that harbor Mycoplasma DNA [94]. Another study reported a rate of only 13% (6 samples out of a total of 46) of mycoplasmas in ovarian cancerous tissue using the nested PCR technique. Sequencing and BLAST analysis of the six positive cases identified M. salivarium and M. arginini. Given the low detection rate and the contaminating nature of the identified Mycoplasma species, the authors stated that their results are insufficiently consistent to confirm any relationship between Mycoplasma and ovarian cancer [95]. Idahl et al. undertook a similar work, but focused on searching for a particular species of Mycoplasma, M. genitalium. eir results revealed the presence of antibodies to M. genitalium in patients with ovarian cancer. But the statistics and comparison with controls showed that the association between this Mycoplasma species and ovarian cancer is not significant [96]. Overall, Mycoplasma detection rates in women with ovarian cancer do not seem to support the hypothesis that these microorganisms are involved in ovarian cancer. Larger epidemiological studies and more sophisticated methods are needed to better define the role of mycoplasmas in the tumorigenesis of ovarian tissue.

Cervical Cancer.
Cervical cancer is one of the most common cancers among women worldwide, particularly in the low-and middle-income countries. Together with breast cancer, it is classified at the top of the most commonly diagnosed cancers and the leading causes of death in women [1]. Some carcinogenic microorganisms such as the human papillomavirus and the human immunodeficiency virus were identified as a cofactor for the development of cancerous lesions in the uterine cervix [115][116][117]. Because they are commonly detected in the cervical microbiome, mycoplasmas have brought the attention of some scientists who have eventually detected them in women with cervical cancer. Some Mycoplasma species were identified, notably, M. penetrans, M. genitalium, M. hominis, and U. urealyticum [97][98][99].  [90]. A tumor affecting the lymphatic system (non-Hodgkin's lymphoma) was also suspected to be related to Mycoplasma infection. Indeed, M. fermentans was incriminated [100]. e relationship between Mycoplasma infection and renal carcinoma has also been investigated. In two different studies, similar high detection rates, exceeding 80% of mycoplasmas DNA have been found in tissues of cancer patients. Such interesting results prompted the authors to consider the clinical significance of Mycoplasma infection in patients suffering from kidney cancer [101,102]. Likewise, the  [103].

Other
Mycoplasmas have been detected in the oral cavity and in the upper and lower respiratory tracts. Baracaldo et al. reported the case of a man with cancer of the larynx, presenting empyema probably caused by M. salivarium. e suspicion of mycoplasmas occurred following the observation of the growth of small Gram-negative colonies in the pleural fluid collected from that patient. No other bacteria or fungi were detected in the sample. PCR followed by sequencing identified M. salivarium [104]. e same species was also detected in a tumor in a patient with squamous cell carcinoma of the tongue [105]. Another study reported the case of a young woman with a cervical squamous cell carcinoma probably related to oropharyngeal infection by M. hominis species [106]. Moreover, patients with lung cancer were also found to harbor mycoplasmas.

Conclusion
Several studies have raised concern over Mycoplasma-cancer subject from different perspectives. e purpose of this article was to review the experimental and epidemiological studies on the possible association between cancer and mycoplasmas. Despite the multiple studies approving that mycoplasmas are endowed with a malignancy potential and those reporting data about their prevalence in patients with different types of cancer, the etiopathological role of mycoplasmas in tumorigenesis is still controversial and debatable. Indeed, the association between Mycoplasma and cancer has been often put in doubt as some studies did not report sufficient evidences on mycoplasmas' oncogenic properties [118] nor on their ability to induce transformation [119]. Aside of the repetitive detection of M. hyorhinis in gastric cancer, no other obvious specific association may be inferred between a given Mycoplasma species and a particular type of cancer. Nevertheless, the detection of mycoplasmas antibodies remains an efficient mean of monitoring their infection in patients with cancer and could provide evidence of their presence and therefore of their possible involvement. Although PCR amplification is the most useful and direct way to detect mycoplasma DNA, the in situ localization by culture remains the best one to confirm the presence of live and active mycoplasmas in tumor sites. is supports the hypothesis of causality for the involvement of these bacteria in host cells transformation.
We cannot ignore the vulnerability to infectious agents, including mycoplasmas, of an immunodeficient organism subsequent to cancer. To exclude such a case, further investigations should be undertaken to provide strong and irrefutable proof of a causal relationship between mycoplasmas and cancer and to elucidate the mechanisms governing their potential for malignancy. In order to do so, researchers should ask a question: could the cancers associated with infectious agents be avoided if the infection in cause is prevented? If it is shown that reliable diagnosis and early prevention of infection will prevent the development of cancer, this would strengthen the theory of the infectious origin of cancers.

Data Availability
e data used to support the findings of this study are included within the article.

Conflicts of Interest
e authors declare that they have no conflicts of interest.

Authors' Contributions
EY and BBAM conceived and designed the review and wrote the manuscript. EY conducted the database search and data collection. OMSAW and MHAlS drafted the article. All the bibliographical sources included in this review were carefully consulted and approved by all the authors. All authors approved the final version of the manuscript.