BIOMARKERS AND MOLECULES FOR USE IN THE TREATMENT OF CANCER PROGRESSION

FIELD OF THE INVENTION

Embodiments of the present disclosure relate to the field of molecular biology and medicine. More specifically embodiments of the present disclosure are directed towards a method to diagnose cancer progression and to molecules for use in the treatment, delay and amelioration of cancer. In particular, embodiments of the present disclosure relate to novel bacterial nucleic acids and proteins diagnostically and/or prognostically and/or predictively relevant biomarkers and methods related to detection and therapeutic treatment thereof with the purpose of treatment, delay of cancer progression and amelioration of health of a subject in case of cancer.

BACKGROUND OF THE INVENTION

Mycoplasma and Diseases

It is known that Mycoplasma is a genus of bacteria that lacks a cell wall around their cell membrane. In humans, some Mycoplasma species are found as commensal inhabitants, while others have a significant impact on the cellular metabolism and physiology. For example, to overcome their limited metabolic capabilities, they assimilate nutrients from the host, thus contributing to the production of reactive oxygen species (ROS).

Mycoplasma sp. can both be associated with infectious diseases and post infection pathologies, and frequently persist as chronic, asymptomatic infections both in humans and animals ( Rottem S. Interaction of mycoplasmas with host cells. Physiol Rev. 2003;83:417-32). In fact, they can cause a wide variety of diseases, including acute respiratory illness ( Uάhbz A, Martinez-Ramos A, Calixto T, Gonzblez-Matus FJ, Rivera-Tapia JA, Giono S, Gil C, Cedillo L. Animal model of Mycoplasma fermentans respiratory infection. BMC Res Notes. 2013;8:6-9.), genitourinary tract ( Fully JG, Taylor-Robinson D, Cole RM, Rose DL. A newly discovered mycoplasma in the human urogenital tract. Lancet. 1981;1:1288-91.), joint infections ( Kawahito Y, Ichinose S, Sano H, Tsubouchi Y, Kohno M, Yoshikawa T, Tokunaga D, Hojo T, Harasawa R, Nakano T, Matsuda K. Mycoplasma fermentans glycolipid-antigen as a pathogen of rheumatoid arthritis. Biochem Biophys Res Commun. 2008;369:561-6.; Gilroy CB, Keat A, Taylor-Robinson D. The prevalence of Mycoplasma fermentans in patients with inflammatory arthritides. Rheumatology. 2001 ;40: 1355-8.) and neurologic disorders ( Narita M. Pathogenesis of neurologic manifestations of Mycoplasma pneumoniae infection. Pediatr Neurol. 2009;41:159-66.).

It was suggested that Mycoplasma might act as cofactor promoting the progression of HIV infection to AIDS, since they have been detected in the blood and in the urine samples of HIV positive patients (Lo SC, Hayes MM, Kotani H, Pierce PF, Wear DJ, Newton PB 3rd, Tully JG, Shih JW. Adhesion onto and invasion into mammalian cells by Mycoplasma penetrans: a newly isolated mycoplasma from patients with AIDS. Mod Pathol. 1993;6:276-80. ; Blanchard A, Montagnier L. AIDS -associated mycoplasmas. Annu. Rev. Microbiol. 1994;48:687-712.

Regarding the mechanism responsible for their potential pathogenic role, Mycoplasmas do possess neither cell wall, as above explained, nor bacterial modulins such as LPS or peptidoglycans. However, there is evidence to show that membrane-bound lipoproteins present in cell membranes of all Mycoplasma species are capable of activating monocytes-macrophages, lymphocytes or fibroblasts that are cytokine-producing cells (. Razin S, Yogev D, Naot Y. Molecular biology and pathogenicity of mycoplasmas. Microbiol Mol Biol Rev. 1998;62:1094-156.; Shibata K, Hasebe A, Into T, Yamada M, Watanabe T. The N-terminal lipopeptide of a 44-kDa membrane-bound lipoprotein of Mycoplasma salivarium is responsible for the expression of intercellular adhesion molecule-1 on the cell surface of normal human gingival fibroblasts. J Immunol. 2000; 1 (165):6538-44.). Mycoplasmas thus seem to have diverse effects on the activation of B and T cells, on the induction of expression of major histocompatibility complex class I and class II molecules ( Stuart PM, Egan RM, Woodward JG. Characterization of MHC induction by Mycoplasma fermentans (incognitus strain). Cell Immunol. 1993; 152:261-70.), on the cytotoxic activity of T cells and natural killer cells ( D’Orazio JA, Cole BC, Stein-Streilein J. Mycoplasma arthritidis mitogen up-regulates human NK cell activity. Infect Immun. 1996;64:441-7.). Moreover, it has been observed an altered cytokines secretion regulation (such as interleukin- 1 (IL-l), IL-2, IL-4, IL-6, interferons, tumor necrosis factor alpha (TNF-a), granulocyte-macrophage colony stimulating factor (GM-CSF) secretion, adhesion molecules expression, transcription factors expression, MAP kinases and apoptotic pathways activities ( Razin S, Yogev D, Naot Y. Molecular biology and pathogenicity of mycoplasmas. Microbiol Mol Biol Rev. 1998;62:1094-156.). Furthermore, the components of the plasma membranes of Mycoplasmas act as agonists of the heterodimeric Toll- like receptors 2 and 6 (TLR2 and TLR6). Signaling through TLR2/6 in turn results in activation of nuclear factor NF-kB, a major mediator of inflammatory responses and anti-apoptotic factor (Takeda et al., 2002).

All these effects could eventually lead to altered immune-system response, though additional studies are on-going to better clarify this issue.

Recently, many reports have strongly suggested a role for Mycoplasma in cellular transformation and the search for the link between Mycoplasma and cancer is currently actively being investigated {Zhang S, Tsai S, Lo SC. Alteration of gene expression profiles during mycoplasma-induced malignant cell transformation. BMC Cancer. 2006;4(6):116.; Afriat R, Horowitz S, Priel E. Mycoplasma fermentans inhibits the activity of cellular DNA topoisomerase I by activation of PARP1 and alters the efficacy of its anti-cancer inhibitor. PLoS ONE. 2013;27(8):e72377. doi: 10.1371/iournal.pone.0072377. ). To this regard, many studies demonstrated the effects of Mycoplasma on cell lines by showing that Mycoplasma may facilitate tumorigenesis, for example in bronchial epithelial cells ( Reddel RR, Salghetti SE, Willey JC, Ohnuki Y, Ke Y, Gerwin BI, Lechner JF, Harris CC. Development of tumor igenicity in simian virus 40- immortalized human bronchial epithelial cell lines. Cancer Res. 1993;53:985- 91.), in hepatocytes {Choi MS, Lee HM, Kim WT, Kim MK, Chang HJ, Lee HR, Joh JW, Kim DS, Ryu CJ. Detection of mycoplasma infection in circulating tumor cells in patients with hepatocellular carcinoma. Biochem Biophys Res Commun. 2014;4(446):620-5.), in oral tissues {Patil S, Rao RS, Raj AT. Role of Mycoplasma in the initiation and progression of oral cancer. J Int Oral Health. 2015;7:i-ii.), in human prostate cells {Namiki K, Goodison S, Porvasnik S, Allan R, Iczkowski K, Urbanek C, Reyes L, Sakamoto N, Rosser C. Persistent exposure to Mycoplasma induces malignant transformation of human prostate cells. PLoS ONE. 2009;4:e6872. doi: 10.1371/jo urnal.pone.0006872. ; Barykova YA, Logunov DY, Shmarov MM, Vinarov AZ, Fiev DN, Vinarova NA, Rakovskaya IV, Baker PS, Shyshynova I, Stephenson AJ, Klein EA, Naroditsky BS, Gintsburg AL, Gudkov A V Association of Mycoplasma hominis infection with prostate cancer. Oncotarget. 2011;2:289-97.) and cervical cells ( Zhang S, Wear DJ, Lo S. Mycoplasmal infections alter gene expression in cultured human prostatic and cervical epithelial cells. FEMS Immunol Med Microbiol. 2000;27:43-50.).

In addition, Mycoplasma fermentans, Mycoplasma penetrans and Mycoplasma hyorhinis were reported to have oncogenic potential. In fact, long-term Mycoplasma infections in cell cultures are associated with increased frequency of chromosomal instability and malignant transformation. They demonstrated not just accumulation of abnormalities but also phenotypic changes of the cells (Tsai S, Wear DJ, Shih JW, Lo SC. Mycoplasmas and oncogenesis: persistent infection and multistage malignant transformation. Proc Natl Acad Sci USA. 1995; 24(92): 10197-201.; Feng SH, Tsai S, Rodriguez J, Lo SC. Mycoplasmal infections prevent apoptosis and induce malignant transformation of interleukin- 3-dependent 32D hematopoietic cells. Mol Cell Biol. 1999; 19:7995-8002. ; Cimolai N. Do mycoplasmas cause human cancer? Can J Microbiol. 2001;47:691-7.). Tsai et al. showed different changes on the cultured cells such as the lost cell-to-cell contact, the spindle morphology and the growth in multiple layers. They demonstrated the reversible nature of these changes by showing that earlier cultured cells (maintained for up to six passages in vitro) treated with three cycles of ciprofloxacin reverted to their original morphology and growth pattern (flat monolayer growth). Instead, cells cultured for more than 18 passages failed to revert to their previous morphology/growth pattern when treated with the same antibiotic, indicating an irreversible change. This indicated that persistent infection with Mycoplasma induced a multistep step carcinogenesis (Tsai S, Wear DJ, Shih JW, Lo SC. Mycoplasmas and oncogenesis: persistent infection and multistage malignant transformation. Proc Natl Acad Sci USA. 1995;24(92):10197-20L). Furthermore long-term infection with Mycoplasma fermentans or Mycoplasma penetrans was reported to induce spontaneous transformation of mouse embryo fibroblasts with overexpression of the H-ras and c-myc proto-oncogenes (Zhang B, Shih JW, Wear DJ, Tsai S, Lo SC. High-level expression of H-ras and c-myc oncogenes in mycoplasma-mediated malignant cell transformation. Proc Soc Exp Biol Med. 1997;14:359-66.).

Logunov and colleagues also showed the effects of Mycoplasma on apoptotic regulators like p53 and NF-KB.

In particular regarding p53, it is known that most tumors are characterized by impairment of p53 pathway, either by mutation of the p53 gene (TP53) (Sous i, T.. p53 alterations in human cancer: more questions than answers. Oncogene, 2007, 26:2145-56), or by deregulation of other components of the pathway (Vogelstein, B., Lane, D., Levine, A.J .. Surfing the p53 network. Nature, 2000, 408(6810): 307 -10). The importance of p53 function as a tumor suppressor is underlined by the fact that at least 50% of human tumors carry mutations in TP53. The determinant role of p53 as tumor suppressor is related to the fact that p53 is a transcription factor that, in response to stress signals, becomes activated and determines different cellular outcomes, as temporary growth arrest and DNA repair, irreversible growth arrest or apoptosis. Furthermore, p53 activity is tightly regulated by several coordinated mechanisms that ensure proper activation including post-translational modifications, as well as interaction with protein partners that modulate its function ( Vogelstein , B. et al. 2000, ref. cit.).

Logunov and colleagues demonstrated reduced activation of p53 with a constitutive activation of NF-kB in cells infected with Mycoplasma, though the responsible Mycoplasma protein was not identified. This altered expression was consistent with many human tumors. Thus, infected cells could evade apoptosis by inhibiting p53 (Logunov DY, Scheblyakov DV, Zubkova OV, Shmarov MM, Rakovskaya IV, Gurova KV, Tararova ND, Burdelya LG, Naroditsky BS, Ginzburg AL, Gudkov A V. Mycoplasma infection suppresses p53, activates NF- kappaB and cooperates with oncogenic Ras in rodent fibroblast transformation. Oncogene. 2008;27:4521-31.).

However, despite these observations in vitro, no carcinogenic role for any Mycoplasma has been demonstrated in vivo.

Moreover, several studies reported the isolation of Mycoplasma species in various infectious, neoplastic tissues and body fluids. Indeed My coplasmas have been found in precancerous lesions as well as in malignant tissues from patients with stomach, colon, ovarian and lung cancers (Chang AH, Parsonnet J. Role of bacteria in oncogenesis. Clin Microbiol Rev. 2010;23:837-57. dot: 10.1128/CMR.00012-10. ).

The involvement of changes in DNA methylation in the process of carcinogenesis is being actively investigated. DNA methylation (that is the conversion of cytosine to 5-methylcytosine) is an essential element in transcriptional regulation and is one of the major epigenetic mechanisms. Many stresses or DNA damage can in fact interfere with the ability of DNA to be methylated at CpG dinucleotides by DNA-methyltransferases (DNA-MTases) (Wachsman, 1997). To better study these events, specific Mycoplasma MTases have been expressed in human cell lines and their translocation to the nucleus has been observed, where these mycoplasmic enzymes methylated cytosines within the respective CG and GATC sites in human genomic DNA. The result was a change of the human genome methylation landscape, together with the stimulation of pro-oncogenic pathways ( Chernov A V, Reyes L, Xu Z, Gonzalez B, Golovko G, Peterson S, Perucho M, Fofanov Y, Strongin AY. Mycoplasma CG- and GATC-specific DNA methyltransferases selectively and efficiently methylate the host genome and alter the epigenetic landscape in human cells. Epigenetics. 2015;10:303-18.).

All these studies and properties of Mycoplasmas suggest that these agents act as cancer-promoting factors.

Chlamydia trachomatis is a very common kind of bacteria that can infect the female reproductive system as well as other parts of the body in both men and women. It is spread through sex.

Although infection of the reproductive organs may cause symptoms in some people, most women have no symptoms. This means that women with chlamydia usually do not know they are infected unless samples are taken during a pelvic exam and tested for chlamydia. It is a common infection in younger women who are sexually active, and can remain for years unless it is detected and treated.

Some studies have found that women whose blood tests showed past or current chlamydia infection may be at greater risk for cervical cancer than women with negative blood test results.

Studies have not shown that chlamydia itself causes cancer, but it might work with HPV (Human Papillomavirus) in a way that promotes cancer growth. For example, researchers have found that women who had chlamydia along with HPV are more likely to still have HPV when they are re-tested later than women who have not had chlamydia. Although more studies are needed to confirm these findings, there are already good reasons to be checked for chlamydia infection and have it treated with antibiotics if it is found.

In women, long-term chlamydia infection is known to cause pelvic inflammation that can lead to infertility, mainly by building up scar tissue in the Fallopian tubes. Like other infections that inflame or cause ulcers in the genital area, chlamydia can also increase the risk of becoming infected with HIV during exposure to an HIV-infected sexual partner.

Bacteria and Cancer

Bacterial infections traditionally have not been considered major causes of cancer. Recently, however, bacteria have been linked to cancer by two mechanisms: induction of chronic inflammation and production of carcinogenic bacterial metabolites. The most specific example of the inflammatory mechanism of carcinogenesis is Helicobacter pylori infection. About 2 in 3 adults worldwide are infected with H pylori. The rate of infection is higher developing countries and in older age groups. It is likely spread in a couple of ways. One is the fecal- oral route, such as through contaminated food or water sources. It can also be transmitted from one person to another, mouth to mouth. Other factors also play a role in whether or not someone develops stomach cancer. For example, nitrites are substances commonly found in cured meats, some drinking water, and certain vegetables. They can be converted by certain bacteria, such as H pylori, into compounds that have been found to cause stomach cancer in lab animals. Antibiotics and other medicines can be used to treat H pylori infections. According to the CDC, people who have active ulcers or a history of ulcers should be tested for H pylori , and, if they are infected, should be treated. Testing for and treating H pylori infection is also recommended after removal of an early stomach cancer.

The following is a list of carcinogenic and suspected carcinogenic bacteria: Bacteroides fragilis, Helicobacter pylori, Helicobacter bilis, Helicobacter hepaticus, Chlamydia trachomatis, Neisseria gonorrhoeae, Propionibacterium acnes, Chlamydophila psittacii, Salmonella enterica serovar Typhimurium, Salmonella enterica serovar Paratyphi, Salmonella enterica serovar Typhim, Mycoplasma ulcerans, Clostridium ssp, Treponema pallidum syphilis, Borrelia burgdorferi, Helicobacter feli, Helicobacter salmonis, Helicobacter bizzozeronii, Helicobacter heilmannii, H. Epaticus, Campylobacter jejuni, Chlamydia pneumonia, Chlamydia psittaci, Mycoplasma sp., Mycoplasma fermentans, Mycoplasma penetrans, Mycoplasma hyorhinis, Streptococcus bovis, Streptococcus mitis, Streptococcus gallolyticus, Staphylococcus epidermis, Bacillus sp., Robinsoniella peoriensis, Pedioccoccus acidilactici, Leuconostoc lactis, L. Mesenteroides, Ralstonia insidiosa, Fusobacterium naviforme, Fusobacterium nucleatum, F necrophorum, F. Mortiferum, F. Perfoetens, Roseburia sp., Faecalibacterium sp., Escherichia coli, Citrobacter sp., Prevotella sp., Fusobacterium sp..

There is therefore a need to improve a method to diagnose cancer progression and molecules for use in the treatment of cancer progression, which overcome at least one of the drawbacks in the art.

Various limitations and disadvantages of conventional solutions and technologies will become apparent to one of skill in the art after reviewing the remainder of the present application with reference to the drawings and description of the embodiments which follow, though it should be understood that this description of the related art section is not intended to serve as an admission that the described subject matter is prior art.

SUMMARY OF THE INVENTION

According to embodiments, a method to diagnose cancer progression in a patient is provided. In one embodiment, the method comprises detecting one or more prokaryotic biomarkers in a biological sample, wherein said prokaryotic biomarkers are derived from the presence of prokaryotic pathogens responsible for cancer progression, further wherein upon detection of said one or more biomarkers a diagnosis of cancer progression is made.

According to possible embodiments, combinable with all embodiments described herein, said one or more biomarkers derived from bacterial presence are selected among: nucleic acids derived from bacterial infection, antigens, antibodies recognizing bacterial antigens, autoantibodies developed against cellular factors raised by the organism in response to bacterial infection.

According to further embodiments, a kit to diagnose cancer progression in a patient by detecting one or more prokaryotic biomarkers in a biological sample is provided, wherein said prokaryotic biomarkers are derived from the presence of prokaryotic pathogens responsible for cancer progression. In one embodiment, said kit comprises and apparatus and/or device configured for performing a detection analysis or test chosen among: protein and nucleid acid nanoswitch- based homogeneous assay; ELISA or other types of immunoassay, chemiluminescence hybridation assay, electrode based immunosensor, Luminex assay, Surface Plasmon Resonance (SPR) assay, Polymerase Chain Reaction (PCR) based test, whereby upon detection of said one or more biomarkers a diagnosis of cancer progression is made.

According to still further embodiments, a molecule for use in the treatment of cancer progression in a patient is provided. In one embodiment, the molecule is a molecule for treatment of prokaryotic pathogens responsible for cancer progression in the patient.

According to possible embodiments, combinable with all embodiments described herein, said molecule is chosen among: RNA inhibitors, protein inhibitors, biopharmaceutical drugs, such as monoclonal antibodies, antibiotics, synthetic peptides, aptamers, inorganic compounds, synthetically engineered vectors, such as viral vectors, RNA interference, or combination thereof.

According to further aspects, a therapeutic kit for use in the treatment of cancer progression in a patient is provided, said kit comprising a molecule according to the present disclosure.

These and other features, aspects and advantages of the present disclosure will become better understood with reference to the following description, the drawings and appended claims. The drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments of the present subject matter and, together with the description, serve to explain the principles of the disclosure.

The various aspects and features described in the present disclosure can be applied, individually, wherever possible. These individual aspects, for instance the aspects and features described in the attached dependent claims, can be made subject of divisional patent applications.

It is noted that anything found to be already known during the patenting process is understood not to be claimed and to be the subject of a disclaimer.

BRIEF DESCRIPTION OF THE DRAWINGS

So that the manner in which the above recited features of the present invention can be understood in detail, a more particular description of the invention, briefly summarized above, may be had by reference to embodiments. The accompanying drawings relate to embodiments of the disclosure and are described in the following:

- Figure 1 is a graph plotting age of mice in weeks vs. % mice developing tumor, showing tumorigenesis in SCID mice by Mycoplasma infection;

- Figure 2 shows the result of a Co-immunoprecipitation of p53 and Mycoplasma DNA-K assay.

DETAILED DESCRIPTION OF THE EMBODIMENTS

Reference will now be made in detail to the various embodiments of the invention, one or more examples of which are illustrated in the figures. Within the following description of the drawings, the same reference numbers refer to the same components. Generally, only the differences with respect to individual embodiments are described. Each example is provided by way of explanation of the invention and is not meant as a limitation of the invention. For example, features illustrated or described as part of one embodiment can be used on or in conjunction with other embodiments to yield yet a further embodiment. It is intended that the present invention includes such modifications and variations.

It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.

Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.

Before describing these embodiments, it shall be also clarified that the present description is not limited in its application to details of the construction and disposition of the components as described in the following description using the attached drawings. The present description can provide other embodiments and can be obtained or executed in various other ways. It shall also be clarified that the phraseology and terminology used here is for the purposes of description only, and cannot be considered as limitative.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, representative illustrative methods and materials are now described.

All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. To the extent such publications may set out definitions of a term that conflicts with the explicit or implicit definition of the present disclosure, the definition of the present disclosure controls. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

Some aspects of the present disclosure relate to novel diagnostically and therapeutically relevant prokaryotic biomarkers related to cancer progression and methods for the detection and treatment of tumorigenic prokaiyotic pathogens.

Other aspects of the present disclosure relate to methods to diagnose and treat prokaryotic pathogens responsible for cancer progression.

Further aspects of the present disclosure relate to novel methods to detect diagnostically and therapeutically relevant prokaryotic biomarkers for cancer progression and treatment for associated pathogens.

Still further aspects of the present disclosure relate to novel diagnostic and therapeutic prokaryotic targets and methods to diagnose and treat pathogens related to cancer progression.

Yet further aspects of the present disclosure relate to novel diagnostic and therapeutic prokaryotic biomarkers and methods to diagnose and treat cancer progression.

Bacteria have been linked to cancer by several mechanisms: they might play a direct causative role, for instance by induction of chronic inflammation and production of carcinogenic bacterial metabolites and therefore by triggering tumorigenesis as a result of bacterial presence within healthy tissue.

On the other side, bacterial infection might arise from spontaneous infection of established tumors, through an opportunistic mechanism.

In both cases, associations between different bacteria and various tumors have been reported in patients for decades.

However, despite the last findings, there is a lack of molecular mechanisms that allow to characterize bacterial cancer-related diagnostic and prognostic biomarkers also useful as therapeutic targets. Embodiments of the present disclosure, therefore, relate to the application of novel bacterial protein and nucleic acid as diagnostic/prognostic biomarkers for cancer diagnosis and progression.

In particular, embodiments described herein relate to a method to diagnose cancer progression in a patient by detecting prokaryotic biomarker(s) in a biological sample, wherein said prokaryotic biomarker(s) are derived from the presence in said sample of prokaryotic pathogens responsible for cancer progression, wherein upon detection of said prokaryotic biomarker(s) a diagnosis of cancer progression is made.

Further embodiments relate to molecules for use in the treatment of cancer progression in a patient, wherein said molecules are molecules for treatment of prokaryotic pathogens responsible for cancer progression in the patient.

In possible embodiments, biomarkers derived from bacterial presence can be selected among the following biomarkers: - nucleic acids derived from bacterial infection,

- antigens (e.g. proteins, peptides, or mixture of proteins, mixture of peptides, or recombinant protein containing multiple epitopes, or synthetic polypeptides containing multiple peptides),

- antibodies recognizing bacterial antigens,

- autoantibodies developed against cellular factors raised by the organism in response to bacterial infection.

Some particular embodiments of the present disclosure relate to nucleic acid traces of a Mycoplasma fermentans strain which has been initially isolated from HIV-positive patients. For the first time, lateral gene transfer of genomic materials belonging to some bacterial strains has been detected in a small percentage of cancer cells and a molecular mechanism that lead to p53 inhibition by some bacterial proteins has been characterized; this discovery implicates new molecular DNA fingerprints which allow novel diagnostic and prognostic evaluations.

The present disclosure, thus, further relates to the use some specific bacterial protein biomarkers as antigenic determinants for the detection of specific antibodies in eukaryotic biological fluids.

The present disclosure further relates to antibodies against transforming bacterial epitopes for vaccines and peptides suitable for tumorigenic pathogens immunotherapies. In some specific embodiments therapeutic molecules against bacteria, transforming protein and RNA are disclosed.

In some embodiments, detection of the prokaryotic biomarker(s) can be perfomed by using one of the following assays or techniques, according to specific needs:

- Protein and Nucleid acid Nanoswitch-based homogeneous assay, as described for instance in international applications WO-A-2015/114506, PCT/EP2017/067904, PCT/IT2017/000233 in the name of the present Applicant;

- ELISA or other types of immunoassay,

- Chemiluminescence hybridation assay,

- Electrode based immunosensor, comprising: capacitive immunosensor, resistance-based biosensor, amperometric immunosensor, voltammetric biosensor, Surface Plasmon Resonance analysis, electrochemiluminescence detection, as described for instance in international application WO-A- 2012/101498 in the name of the present Applicant,

- Luminex assay,

- Surface Plasmon Resonance assay,

- Polymerase Chain Reaction (PCR) based test, as described for instance in international applications PCT/IT2017/000125 and PCT/IT2017/000126 in the name of the present Applicant.

In some embodiments, novel bacterial nucleic acids and proteins can be used as diagnostically and/or prognostically and predictively relevant biomarkers related to cancer progression; the detection of such biomarkers can be achieved for instance through the abovementioned assays or techniques, according to specific needs.

In possible embodiments, combinable with all embodiments described herein, the sample that is subjected to detection in the method of the present disclosure can be any biological sample taken from a patient, in particular from humans. The diagnostic method of the present disclosure, therefore, is not practised on the body of the patient but on a sample taken from the patient. The sample is preferably a sample that can be analysed and subjected to one of the above mentioned detection tests or assays. Possible samples that can be used in connection with the embodiments described herein include: blood (e.g. whole blood, blood plasma, serum, possibly with anticoagulant), urine, feces, stools extracts, cerebrospinal fluid (CSF), tissues or cells biopsies, kidney stones, aspirated samples (e.g. bone marrow, pleura fluid, synovial fluid, intestinal fluid), bodily fluids (such as mucus, saliva, sweat,), sebaceous secretions, liquid phase of cell and tissue extracts, tissues, biopsies, needle-aspirated samples.

Nucleic Acids

According to embodiments, nucleic acids derived from bacterial infection can be used as diagnostically and/or prognostically and/or predictively relevant biomarkers related to cancer development and progression in the method of the present disclosure.

In particular embodiments, nucleic acids can be a DNA or a RNA of bacterial origins.

In further particular embodiments, bacterial DNA can be present as episome or plasmids inside the host cells or in the host tumor environment.

In still further particular embodiments, bacterial DNA can be integrated into the eukaryotic cell genome.

In yet further particular embodiments, bacterial DNA can be integrated into the eukaryotic cell genome and the transcribed RNA represents a diagnostic and/or prognostic biomarker.

In still further particular embodiments, bacterial DNA can be integrated into the eukaryotic cell genome and the relative encoded protein represents a diagnostic, prognostic biomarker.

In still further particular embodiments, bacterial DNA can be integrated into the eukaryotic cell genome and the relative antibody developed against the encoded protein represents a diagnostic and/or prognostic biomarker.

In specific embodiments, the present disclosure further relates to bacterial DNA sequences diagnostically and/or prognostically and/or predictively relevant for cancerogenic transformation. The presence of these sequences in cancer cells can represent a risk factor for tumor transformation.

In some particular embodiments, DNA derived from Mycoplasma sp. can be present in the tumor environment or in the host cells as episome or plasmidic form.

In some particular embodiments, DNA derived from Mycoplasma sp. can be integrated into the eukaryotic genome.

In some particular embodiments, specific sequences of bacterial DNA can be integrated into the eukaryotic genome.

In some particular embodiments, specific sequences of DNA derived from Mycoplasma sp. can be integrated into the eukaryotic genome.

In some particular embodiments, specific sequences of DNA derived from Mycoplasma fermentans can be integrated into the eukaryotic genome.

In some particular embodiments, sequences of DNA-K gene of bacterial origin can be completely or partially integrated into the eukaryotic genome.

In some particular embodiments, sequences of DNA-K gene derived from Mycoplasma sp. can be completely or partially integrated into the eukaiyotic genome (SEQ ID No.: 1).

In some particular embodiments, sequences of DNA-K gene derived from Mycoplasma fermentans can be completely or partially integrated into the eukaryotic genome.

In some particular embodiments, sequences of other genes derived from Mycoplasma fermentans can be completely or partially integrated into the eukaryotic genome (SEQ ID No.: 2-12).

According to embodiments in which a nucleic acid derived from bacterial infection, e.g. a bacterial DNA, is used as biomarker in the method of the present disclosure, the detection of the nucleic acid may occur via the following methods, according to needs and according to the sample that is subjected to analysis: PCR, Real Time PCR, PCR coupled with HRM methods, biosensors for the detection of nucleic acids, immunoassays for detection of DNA, sequencing.

According to embodiments, PCR-based assays can be based on the primers of the present disclosure according to sequences SEQ ID No.: 13-18.

According to embodiments, PCR-based assays can be based on other primers designed on the basis of the sequences SEQ ID No.: 2-12.

According to embodiments, PCR-based assays can be based on other primers designed on the basis of the homologues of sequences SEQ ID No.: 2-12, belonging to other bacterial strains.

Antigens

According to further embodiments, antigens from bacterial infection can be used as diagnostically and/or prognostically and/or predictively relevant biomarkers related to cancer development and progression, in the method of the present disclosure.

In particular embodiments, antigen can be a bacterial protein.

Bacterial proteins used in the embodiments described herein can be bacterial proteins that interact with cellular proteins involved in cancer development, and through interaction these bacterial proteins can trigger, favor or enhance cancer development.

In possible embodiments, the involved cellular proteins can be selected among p53 (tumor protein p53) or one of the following oncogenes or onco-suppressors: ABL 1 ;MLLT 10 ; AFDN;MLLT 6 ;ZF YVE 19 ; AKAP 13 ; AKT 1 ; ARAF ; AFF4 ; AGR 2;AKIP 1 ;MLLT 1 1 ;AKT2;MLLT3 ;AFF 1 ;ALK;ARHGEF 5 ;NET 1 ;ASPSCRl ;AR HGEF12;BCL2;AURKA;BCAS3;BCL3;BCAS4;BCL9;BCL6;BRI3BP;BTG1 ;B RAF;BMI 1 ;BRCC3 ;BCR;CCDC6;CDT 1 ;CCND 1 ;CCNL 1 ;CBL;CMC4;CRK;CS NK2 A3 ;CREB3L2;DCUN ID 1 ;CSF 1 R;SPECC 1 ;DEK;DOCK4;DDX6;DDIT3 ;E GFR;ENTPD5;EVI2A;EVI2B;ETS1 ;ETV6;MLLT1 ;ERG;MECOM;EWSRl ;EP S 15;ETS2;ELF4;ELL;FES;FGF3;FGF4;FGF5;FAM83B;FAM83D;FAM83A;FC GR2B;FGF6;FER;FGR;ETV1 ;FLI 1 ;FOXOl FYN;FSTL3;F0X03;FGFR2;F0S; FRAT1 ;GLIl ;FLT3;FOX04;GFI1B;GNAS;GAS7;FUS;HCK;HMGA2;GMPS; MDS2;LYL 1 ;MAS 1 ;MDM2 ;MAF;MAFB ;MAF A;LETMD 1 ;LYN;MAP3K8;CB FB ;PAX5 ;P AX7 ;PCM 1 ;PATZ1 ;PAX3;PDGFB;HLF;HOPX;IL2;HOXA9;JAK2; JUN ;KMT2 A; JAZF 1 ;PRKCI;KAT6A;KDSR;PRKCA;LHX4;KIT;LCK;MKL 1 ; MET ;MERTK;MCF2L;MCF2 ;MOS ;RUNX 1 T 1 ;M YB ;NCOA 1 ;MT CP 1 ;M Y CN ; MYEOV;MYHl 1 ;MYC;NFKB2;MXI 1 ;NPM1 ;NR4A3 ;NCOA4;NTRKl ;NSD 1 ; N SD3 ;NUP214 ;POU2 AF 1 ;OLIG2 ;N SD2 ;PDGFD;PHB ;PBX 1 ;PDGFRB ;PL AG 1 ;PDGFRA;PIC ALM;PIM 1 ;PIM3 ;PIK3 C A;PIM2 ;PML ;PTT G2 ;PRCC ;PTT G3P ;RAB8A;RAF 1 ;KRAS;PTTG 1 ;RARA;RARB;HRAS;NRAS;TAF 15 ;REL;ARH GAP26;RHOA;RBM 15 ;LMO 1 ;LMO2;RET;ELAC2;RNF213 ;RRAS2;ROS 1 ;RU NX1 ;SEC31 A; YAP 1 ; YES 1 ;SET;SKI;SH3GL 1 ;SRC;STIL;STYKl ;SUZ12;SSX 2;SSX2B;SSXl ;SS18;CARS;SPIl ;NCKIPSD;TAL2;TALl ;TOPl ;TBClD3;TCL 1 B ;TCTA;TFG;TCF3 ;TFE3 ;TFPT;TCL 1 A;TNFRSF 17 ;TLX 1 ;TRIM37 ;TRIM27 ;TPM3 ;TPR;U SP4 ;USP6; AXL ; VA V 1 ; WNT 1 ; WNT3 ; WWTR 1 ; WDR 1 1 ; WISP 1 ; ZNF521 ;ZBTB 16;ZNF320;AIM2;APC;AGAP2;ARID3B;PYCARD;CDKN2A; ATM;B ANP; AXIN 1 ;BIN 1 ;BRD7 ;BRCA2;BUB 1 B ;CADM 1 ;BRMS 1 ;CADM4;

B AX;BCL 10;BRC A 1 ;CDC73 ;CDK2 AP 1 ;CDKN 1 B ;CAVIN3 ;CDKN2A;CCAR 2;CHD5;CHEK2;C 10orf99;CDKN 1 C;CDKN2B;CDKN2D;C 10orf90;CTCF ;CR EBL2 ;MCC ;DMTN ;DCC ;DAB2IP;C YLD;DAPK3 ;D AB2 ;DMBT 1 ;DFNA5 ;DL EC 1 ;DEC 1 ;DIS3L2;DMTF 1 ;EFNAl ;DPH 1 ;EPB41L3 ;ERRFI 1 ;EPHB2;EXT2;F ES ;FHIT ;FLCN ;F AM 120 A;EXT 1 ;F AM21 OB ;FH;FRK;LGR6 ;MCTS 1 ;MAF ;M AFB;MAFA;PAF 1 ;PBRM1 ;TP73 ;PALB2;TP53 ;PANO 1 ;PARK7;P21 ;HTATIP2 ;HIC 1 ;HIF3 A;PYHIN1 ;ING1 ;ING4;IRF 1 ;KANK1 ;KLKlO;KCTDl 1 ;KCTD21 ;P RKCD;L ACTB ;LATS 1 ;PRKCI;RPS6KA2;KCTD6;LATS2;LIN9;DLEU1 ;LIM D 1 ;NF2;MFHAS 1 ;MLH1 ;LZTS1 ;MAPKAPK5;MN1 ;MSH2;MTSS 1 ;MTUS 1 ; MUC 1 ;NAT 6 ;NDRG2 ;NBL 1 ;MUTYH;NEURL 1 ;NF 1 ;NKX3- 1 ;NPRL2;GPR68;PDCD4;PHLPPl ;PHLPP2;PHLDA3;PINX1 ;PLK2;PLPP5;PN N;PLEKHG2;HPGD;PLEKHO1 ;PML;PMS 1 ;PMS2;PRR5;PTEN;PTCHl ;RASS F2;RB 1 CC 1 ;GPRC5A;RASA 1 ;RASSF5;RASSF 1 ;RBL 1 ;RAP 1 A;RBL2;RB 1 ;D LC 1 ;RASSF4;RBMX;RECK;RHOB;ARHGAP20;RASL 1 OA;SDHA;SASH 1 ;SL C5A8;SETD2;SIRT4;SIKl;STARDl3;STKl l;SUFU;ST20;SUSD2;SMARCBl ; SUSD6;SYNPO2;TP53INP1 ;TRIM24;TCHP;TET2;TCP10L;TBRG1;TSC2;TS C 1 ;TMEM 127 ;TUSC2;VHL;UFL 1 ; VWA5 A;TXNIP; WT 1 ; WWOX;XAF 1 ;XRN 1 ;ZMYND 11 ;ZBTB7C;ZDHHC 17.

In particular, it has been found that p53 is able to interact with seven proteins: chaperon protein DNA-K, phenylalanine-tRNA ligase alpha subunit, 1-deoxy-D- xylulose-5 -phosphate synthase family protein, ATP synthase FI beta subunit, glyceraldehyde-3 -phosphate dehydrogenase type I, arginine-tRNA ligase, phosphopyruvate hydratase (SEQ ID No.: 20-25). This interaction leads to the deregulation of the protein activity and expression, with consequently effect of neoplasia development. See experimental data discussed hereinbelow with reference to Figures 1 and 2 and regarding p53.

In particular, the protein that can be considered for interaction with p53 in embodiments of the present disclosure can be a bacterial DNA-K protein.

In further embodiments, the above mentioned seven proteins can be derived from Mycoplasma sp., in particular Mycoplasma fermentans.

In still further embodiments, a DNA-K protein deriving from Mycoplasma sp., in particular Mycoplasma fermentans can be used (SEQ ID No.: 19).

In other particular embodiments, antigen can be a peptide derived from a bacterial protein.

In further particular embodiments, antigen can be a mixture of bacterial proteins.

In still further particular embodiments, antigen can be a mixture of peptides derived from a bacterial protein.

In yet further particular embodiments, antigen can be a recombinant protein, artificially designed, containing multiple epitopes corresponding to bacterial proteins.

In still further particular embodiments, antigen can be a recombinant protein, artificially designed, containing multiple epitopes corresponding to bacterial proteins and host proteins.

In yet further particular embodiments, antigen can be synthetic polypeptides containing multiple peptides derived from bacterial proteins.

In still further particular embodiments, antigen can be synthetic polypeptides containing multiple peptides derived from bacterial proteins and host proteins.

In still further particular embodiments, antigen can be derived from one or more of the following bacteria: Bacteroides fragilis, Helicobacter pylori, Helicobacter bilis, Helicobacter hepaticus, Chlamydia trachomatis, Neisseria gonorrhoeae, Propionibacterium acnes, Chlamydophila psittacii, Salmonella enterica serovar Typhimurium, Salmonella enterica serovar Paratyphi, Salmonella enterica serovar Typhim, Mycoplasma ulcerans, Clostridium ssp, Treponema pallidum syphilis, Borrelia burgdorferi, Helicobacter feli, Helicobacter salmonis, Helicobacter bizzozeronii, Helicobacter heilmannii, H. Epaticus, Campylobacter jejuni, Chlamydia pneumonia, Chlamydia psittaci, Mycoplasma sp., Mycoplasma fermentans, Mycoplasma penetrans, Mycoplasma hyorhinis, Streptococcus bovis, Streptococcus mitis, Streptococcus gallolyticus, Staphylococcus epidermis, Bacillus sp., Robinsoniella peoriensis, Pedioccoccus acidilactici, Leuconostoc lactis, L. Mesenteroides, Ralstonia insidiosa, Fusobacterium naviforme, Fusobacterium nucleatum, F necrophorum, F. Mortiferum, F. Perfoetens, Roseburia sp., Faecalibacterium sp., Escherichia coli, Citrobacter sp., Prevotella sp., Fusobacterium sp _¬ in further particular embodiments, antigen can be derived from the DNA-K proteins of one or more of the following bacteria: Bacteroides fragilis, Helicobacter pylori, Helicobacter bilis, Helicobacter hepaticus, Chlamydia trachomatis, Neisseria gonorrhoeae, Propionibacterium acnes, Chlamydophila psittacii, Salmonella enterica serovar Typhimurium, Salmonella enterica serovar Paratyphi, Salmonella enterica serovar Typhim, Mycoplasma ulcerans, Clostridium ssp, Treponema pallidum syphilis, Borrelia burgdorferi, Helicobacter feli, Helicobacter salmonis, Helicobacter bizzozeronii, Helicobacter heilmannii, H. Epaticus, Campylobacter jejuni, Chlamydia pneumonia, Chlamydia psittaci, Mycoplasma sp., Mycoplasma fermentans, Mycoplasma penetrans, Mycoplasma hyorhinis, Streptococcus bovis, Streptococcus mitis, Streptococcus gallolyticus, Staphylococcus epidermis, Bacillus sp., Robinsoniella peoriensis, Pedioccoccus acidilactici, Leuconostoc lactis, L. Mesenteroides, Ralstonia insidiosa, Fusobacterium naviforme, Fusobacterium nucleatum, F necrophorum, F. Mortiferum, F. Perfoetens, Roseburia sp., Faecalibacterium sp., Escherichia coli, Citrobacter sp., Prevotella sp., Fusobacterium sp.

In further embodiments, the bacteria can be Mycoplasma sp. and the antigen can be derived from one of the following proteins of Mycoplasma sp.: chaperon protein DNA-K, phenylalanine-tRNA ligase alpha subunit, 1-deoxy-D-xylulose- 5 -phosphate synthase family protein, ATP synthase FI beta subunit, glyceraldehyde-3 -phosphate dehydrogenase type I, arginine-tRNA ligase, phosphopyruvate hydratase (SEQ ID No.: 20-25).

In further embodiments, antigen can be derived from one of the homologues, present in one of the other bacterial species as above described, of the following proteins of Mycoplasma sp.,: chaperon protein DNA-K, phenylalanine-tRNA ligase alpha subunit, l-deoxy-D-xylulose-5-phosphate synthase family protein, ATP synthase FI beta subunit, glyceraldehyde-3 -phosphate dehydrogenase type I, arginine-tRNA ligase, phosphopyruvate hydratase.

Antibodies

According to further embodiments, antibodies arisen following bacterial infection can be used as diagnostically, prognostically and/or predictively relevant biomarkers related to cancer development and progression, in the method of the present disclosure. Therefore, epitopes recognized by the aforementioned antibodies can be used as bait in diagnostic assays.

In some particular embodiments, antibodies useful for serodiagnosis can be antibodies raised against bacterial antigens according go embodiments described above.

In other particular embodiments, antibodies useful for serodiagnosis can be autoantibodies raised against human antigens when bacterial infection is present, according to embodiments described above.

In further particular embodiments, antibodies can recognize epitopes derived from DNA-K proteins of bacterial origins.

In still further particular embodiments, antibodies can recognize epitopes derived from bacterial proteins that interact with cellular proteins involved in cancer development, triggering, favoring or enhancing cancer development.

According to further embodiments, said cellular protein is selected among: p53 or one of the above-indicated oncogenes or onco-suppressors. In further embodiments, said cellular protein is p53 and the proteins interacting with p53 are selected among: chaperon protein DNA-K, phenylalanine-tRNA ligase alpha subunit, l-deoxy-D-xylulose-5-phosphate synthase family protein, ATP synthase FI beta subunit, glyceraldehyde-3- phosphate dehydrogenase type I, arginine-tRNA ligase, phosphopyruvate hydratase.

According to still further embodiments, the proteins interacting with p53 are proteins of Mycoplasma sp., in particular Mycoplasma fermentans, or homologues, present in one of the other above-indicate bacterial species, of said proteins of Mycoplasma sp..

In yet further particular embodiments, antibodies can recognize epitopes derived from DNA-K proteins of Mycoplasma sp., in particular of Mycoplasma fermentans.

In still further particular embodiments, antibodies can recognize epitopes derived from the following antigens of Mycoplasma sp.: chaperon protein DNA- K, phenylalanine-tRNA ligase alpha subunit, l-deoxy-D-xylulose-5-phosphate synthase family protein, ATP synthase FI beta subunit, glyceraldehyde-3- phosphate dehydrogenase type I, arginine-tRNA ligase, phosphopyruvate hydratase (SEQ. ID No.: 20-25).

In further particular embodiments, antibodies can recognize epitopes derived from the proteins of one or more of the following bacteria: Bacteroides fragilis, Helicobacter pylori, Helicobacter bilis, Helicobacter hepaticus, Chlamydia trachomatis, Neisseria gonorrhoeae, Propionibacterium acnes, Chlamydophila psittacii, Salmonella enterica serovar Typhimurium, Salmonella enterica serovar Paratyphi, Salmonella enterica serovar Typhim, Mycoplasma ulcerans, Clostridium ssp, Treponema pallidum syphilis, Borrelia burgdorferi, Helicobacter feli, Helicobacter salmonis, Helicobacter bizzozeronii, Helicobacter heilmannii, H. Epaticus, Campylobacter jejuni, Chlamydia pneumonia, Chlamydia psittaci, Mycoplasma sp., Mycoplasma fermentans, Mycoplasma penetrans, Mycoplasma hyorhinis, Streptococcus bovis, Streptococcus mitis, Streptococcus gallolyticus, Staphylococcus epidermis, Bacillus sp., Robinsoniella peoriensis, Pedioccoccus acidilactici, Leuconostoc lactis, L. Mesenteroides, Ralstonia insidiosa, Fusobacterium naviforme, Fusobacterium nucleatum, F necrophorum, F. Mortiferum, F. Perfoetens, Roseburia sp., Faecalibacterium sp., Escherichia coli, Citrobacter sp., Prevotella sp., Fusobacterium sp..

In other particular embodiments, antibodies can recognize epitopes derived from the DNA-K proteins of one or more of the following bacteria: Bacteroides fragilis, Helicobacter pylori, Helicobacter bilis, Helicobacter hepaticus, Chlamydia trachomatis, Neisseria gonorrhoeae, Propionibacterium acnes, Chlamydophila psittacii, Salmonella enterica serovar Typhimurium, Salmonella enterica serovar Paratyphi, Salmonella enterica serovar Typhim, Mycoplasma ulcerans, Clostridium ssp, Treponema pallidum syphilis, Borrelia burgdorferi, Helicobacter fell, Helicobacter salmonis, Helicobacter bizzozeronii, Helicobacter heilmannii, H. Epaticus, Campylobacter jejuni, Chlamydia pneumonia, Chlamydia psittaci, Mycoplasma sp., Mycoplasma fermentans, Mycoplasma penetrans, Mycoplasma hyorhinis, Streptococcus bovis, Streptococcus mitis, Streptococcus gallolyticus, Staphylococcus epidermis, Bacillus sp., Robinsoniella peoriensis, Pedioccoccus acidilactici, Leuconostoc lactis, L. Mesenteroides, Ralstonia insidiosa, Fusobacterium naviforme, Fusobacterium nucleatum, F necrophorum, F. Mortiferum, F. Perfoetens, Roseburia sp., Faecalibacterium sp., Escherichia coli, Citrobacter sp., Prevotella sp., Fusobacterium sp..

In further embodiments, antibodies can recognize epitopes derived from the antigens corresponding to the homologues, present in other bacterial species, of the following proteins of Mycoplasma sp.: chaperon protein DNA-K, phenylalanine-tRNA ligase alpha subunit, 1-deoxy-D-xylulose- 5 -phosphate synthase family protein, ATP synthase FI beta subunit, glyceraldehyde-3- phosphate dehydrogenase type I, arginine-tRNA ligase, phosphopyruvate hydratase.

In further embodiments, antibodies can recognize epitopes described in the partial or complete sequences according to any of: SEQ. ID No.: 26-875.

Drugs or Molecules Against Transforming Bacterial Strains and Molecular Cancerogenic Determinants

Further aspects relate to molecules or drugs useful in the therapeutic treatment of cancer. In particular, embodiments described herein concern drugs or molecules for use in the treatment of cancer progression in a patient, wherein said drugs or molecules are molecules for treatment of prokaryotic pathogens responsible for cancer progression in the patient.

More particularly, molecules or drugs for use in immunotherapy of infections by prokaryotic pathogens in order to treat cancer progression in the patient are regarded as being included in the present disclosure.

Therefore, possible embodiments relate to possible therapeutic strategy for curing, treating or inhibiting cancer development and progression, through the eradication, prevention and treatment of the bacterial strains in both causative and opportunistic infections that can be present in a tumor environment.

The therapeutic strategy according to embodiments described herein may include the use of different molecules or drugs, also in combination.

Drugs or molecules that can be used in the embodiments described herein can be selected from a group consisting of: RNA inhibitors, protein inhibitors, biopharmaceutical drugs, such as monoclonal antibodies, antibiotics, synthetic peptides, aptamers, inorganic compounds, synthetically engineered vectors, such as viral vectors, RNA interference, or combination thereof.

In particular embodiment, bacterial DNA can be integrated into the eukaryotic cell genome wherein transcribed RNA contributes to cancer progression and can represent target for specific inhibitors.

In other particular embodiments, RNA inhibitors can be proteins, inorganic compounds, synthetic compounds, that are able to act on RNA synthesis, transport, translation and are selected from a group consisting of: rifampicin, rifamycins, quinolones, fluoroquinolones, antifolates, flucytosines, actinomycin D, doxorubicin, levofloxacin, norfloxacin, ciprofloxacin, irinotecan, etoposide, rifabutin, amphotericin B, mithramycin A, 7-aminoactinmycin D, indazolo- sulfonamide compounds, thiolutin, cystosine analogues, 1-beta-D- arabinofuranosylcytosine, aureothricin, ethidium bromide, 2’-O-methyl guanosine, Acridine orange, rugulosin, alpha-amanitin, juglone, resistomycin, deacetylcolchiceine, 2’C-methyl cytidine, Hetrocyclic inhibitors, rifapentine, foscamet sodium, rubrofusarin and other RNA inhibitor molecules, or a combination thereof.

In further particular embodiments, RNA inhibitors can be based on RNA interference, using sequences that are complementary to the RNA sequences to be degraded. In further particular embodiments, protein inhibitors can target bacterial proteins that are able to contribute to cancer progression.

In other particular embodiments, protein inhibitors can be synthetic peptides.

In further particular embodiments, protein inhibitors can be aptamers.

In still further particular embodiments, protein inhibitors can be inorganic compounds.

In yet further particular embodiments, biopharmaceutical drugs can be used to target bacterial infection that contribute to cancer progression.

In some further particular embodiments, the biopharmaceutical drug used can be a monoclonal antibody.

In still further particular embodiments, the biopharmaceutical drug used can be a RNA molecule.

In yet further particular embodiments, the biopharmaceutical drug used can be based on gene therapy.

In further particular embodiments, the biopharmaceutical drug used can be synthetically engineered vectors, containing a protein, or peptide gene, or a RNA molecule able or configured to treat the bacterial infection, once vector infects the tumoral cells or the cells present in the tumor environment.

In still further particular embodiments, antibiotics that are used to treat bacterial infection can be used to contribute to treat cancer progression according to the present disclosure. Such antibiotics can be selected from a group consisting of: aminoglycosides, ansamycins, carbacephem, geldanamycin, herbimycin, carbapenems, cephalosporins, glycopeptides, lincosamides, lipopeptides, macrolides, monobactams, nitrofurans, oxazolidinones, penicillins, polypeptides, quinolones, fluoroquinolones, sulfonamides, tetracyclines, azithromycin, clarithromycin, erythromycin, roxithromycin, telithromycin, spiramycin, ciprofloxacin, enoxacin, gatifloxacin, gemifloxacin, levofloxacin, lomefloxacin, moxifloxacin, nalidixic acid, norfloxacin, demeclocycline, doxycycline, minocycline, oxytetracycline, tetracycline, 6-thioguanine, gemcitabine, ribavirin, petoxifylline, ganciclovir, zidovudine, stavudine, acyclovir, trifluorothymidine, 5 -iododeoxy uridine, 5-fluorodeoxyuridine, cidofovir, 5-fluorouracil, 5- fluorocytosine, dipyridamole, gemcitabine or combination thereof.

EXPERIMENTAL DATA Experiments showing induction of lymphomas in SCID mice

A strain of M. fermentans was tested in a severe combined immune-deficient (SCID) mouse model ( Prkdc ^A ).

These mice have a defect in DNA repair caused by the lack of DNA-PK. B and T cells do not mature because of the inability to recombine immunoglobulin and T cell receptor chains, respectively, thus these lymphocytes cannot progress through the cell cycle and this eventually leads to their p53-dependent apoptosis.

For this reason, these animals lack B and T cells, although not completely, since some immature cells develop, particularly in the T cell lineage. SCID mice develop T-cell lymphoma (about 40-60% within 40-50 weeks of age). SCID ^Prkdc - ⁱ - /pjj- ^t - d _evei0 p c _ce ll lymphomas at a faster rate (by about 14 weeks of age), indicating that p53 provides a protective effect.

Non-obese diabetic (NOD)/SCID were infected with Mycoplasma to test the hypothesis that this mycoplasma would accelerate lymphoma by interacting with p53 in vivo.

As a negative control, NOD.Cg -Prkdc ^sc,d Il2rg ^tmIWjl /SzJ mice were used, also known as NOD/SCID Gamma (NSG), which do not express the prkdc gene or the X-linked IL-2R gene . These animals do not develop spontaneous T-cell lymphoma even after sub-lethal irradiation, most likely because the lack of a functional IL-2 receptor further hampers T-cell proliferation.

Uninfected controls and infected NSG mice did not develop tumors during the time of the experiment (see Fig. 1).

However, following MF-I infection of the SCID mice, enlarged spleens, thymuses and lymph nodes were apparent as early as 8 weeks after infection.

Histochemical analyses showed lymphoid cells infiltrating the organs of infected animals.

To verify that infiltrating lymphocytes causing organ enlargement were transformed, aliquots of single-cell suspensions from an enlarged lymph node of an MF-I infected animal were injected intra-peritoneally into young (4-8 weeks old) NOD/SCID mice. Extra-nodal tumors were detected as early as 2 weeks after injection. Secondary tumor cells were phenotypically characterized by flow cytometry as CD4 ⁺ /CD8 ⁺ CD3 ^high and CD4 ⁺ /CD8 ⁺ CD3 cells.

Inverted Kap an-Meyer formula was used to generate a plot of the time to tumor development. CB 17.SCID (N=23) and NOD/SCID (N=20) mice were infected with mycoplasma. The experiments were carried out for about 18 weeks after infection, until the animals reached an age of about 25 weeks. A total of 19 animals (10 CB17.SCID and 9 NOD/SCID) developed tumors out of 43 infected. The animals belonged to a colony maintained in animal facility of the Applicant, under pathogen-free conditions. Control, uninfected CB17.SCID mice had a lifespan of about 40-50 weeks, while NOD/SCID mice had a lifespan of 38-45 weeks. Spontaneous T-cell lymphoma was observed in about 30% of the CB17.SCID animals after 33 weeks of age, and in more than 80% of the NOD/SCID animals after 30 weeks of age. Young animals (4-8 weeks old) were infected by intra-peritoneal (i.p.) injection with mycoplasma. Tumor development was observed in animals infected with mycoplasma grown in either aerobic or anaerobic conditions. As early as 7 weeks post infection (p.i.), the spleen and lymph nodes were enlarged in animals infected with mycoplasma. In some animals tumor cells colonized the vestigial thymic area, and autopsy showed an enlarged tumor mass. About 25% of the animals died of wasting within 30 weeks of infection. Age-matched uninfected CB17.SCID (N=10) and NOD/SCID (N=10) animals were kept in adjacent cages as controls. Only 1 NOD/SCID developed a spontaneous tumor at 26 weeks of age. As a further control, we used NSG mice, which are resistant to lymphoma development even after sub-lethal irradiation treatment. None of the infected NSG animals (N=10) developed tumors during the time of the experiment. When SCID animals (N=20) were infected with the prototype M. fermentans PG18 strain, about 80% died of wasting and none developed lymphoma within 36 weeks after infection. This would indicate a more potent immunoreaction of the animals to pg!8 infection leading to death before the development of lymphoma.

Fig. 2 shows the result of co-immunoprecipitation of p53 and Mycoplasma DNA-K. Following transfection and treatment with 5-FU, total proteins from a lysate of HCT116 p53+/+ were immuno-precipitated with anti-p53 mAb or anti- V5 (which recognizes the DNA-K tag), then analyzed by western blot using the appropriate antibody. V5-DNA-K: tag sequence for DNA-K.

To verify interaction of p53 with DNA-K, the Applicant performed immuno- precipitation analysis. A lysate from 1 ^c 10 HCT116 p53 cells transfected with DNA-K or with the vector control was prepared in 0.40 ml of radioimmune precipitation assay solution (cell signaling). Cell lysate was first mixed with 2 pg of preimmune mouse IgG (Sigma- Aldrich) and 30 mΐ of protein G plus/protein A- agarose (Calbiochem) and incubated for 2 h at 4°C with end-over-end rotation. Agarose beads and nonspecifically bound proteins were removed by centrifugation at 10,000 rpm for 10 min at 4°C. The supernatant was then mixed with protein G plus/protein A-agarose (30 mΐ) that had been preincubated with 2 pg of mouse anti-p53 mAb (Cell signaling) or mouse mAb against V5 (Invitrogen) and incubated for 4 h or 16 h at 4°C with end-over-end rotation. The beads and bound proteins were collected by centrifugation at 10,000 rpm for 10 min at 4°C, washed 3 times with radioimmune precipitation assay solution, and resuspended in SDS-PAGE sample buffer. 1/20 of the total immuno-precipitated product was analyzed by SDS-PAGE immunoblot as described above.

For western blot analysis, cell monolayers were detached by scraping, washed in cold PBS and solubilized in Ripa lysis buffer (Sigma) in the presence of protease inhibitors (Sigma). The amount of extracted protein was measured by the Bradford assay (Bio-Rad). 30pg of proteins were resolved by SDS-page, transferred to polyvinylidene difluoride membrane (Bio-Rad), and probed with anti-p53 (Santa Cruz), anti-p21 (Abeam), anti-Bax (Cell Signaling), anti-PUMA (Calbiochem), anti-V5 (Invitrogen), and anti-beta actin (Cell Signaling) antibodies. Blots were incubated with a secondary horseradish peroxidase (HRP)- conjugated antibody (Santa Cruz), developed using an ECL chemiluminescent substrate kit (Amersham Bioscience) and exposed to Kodak x-ray film.

While the foregoing is directed to embodiments of the invention, other and further embodiments of the invention may be devised without departing from the basic scope thereof, and the scope thereof is determined by the claims that follow.