Increased virulence of the oral microbiome in oral squamous cell carcinoma revealed by metatranscriptome analyses

Abstract

Oral squamous cell carcinoma (OSCC) is the most prevalent and most commonly studied oral cancer. However, there is a void regarding the role that the oral microbiome may play in OSCC. Although the relationship between microbial community composition and OSCC has been thoroughly investigated, microbial profiles of the human microbiome in cancer are understudied. Here we performed a small pilot study of community-wide metatranscriptome analysis to profile mRNA expression in the entire oral microbiome in OSCC to reveal molecular functions associated with this disease. Fusobacteria showed a statistically significantly higher number of transcripts at tumour sites and tumour-adjacent sites of cancer patients compared to the healthy controls analysed. Regardless of the community composition, specific metabolic signatures were consistently found in disease. Activities such as iron ion transport, tryptophanase activity, peptidase activities and superoxide dismutase were over-represented in tumour and tumour-adjacent samples when compared to the healthy controls. The expression of putative virulence factors in the oral communities associated with OSCC showed that activities related to capsule biosynthesis, flagellum synthesis and assembly, chemotaxis, iron transport, haemolysins and adhesins were upregulated at tumour sites. Moreover, activities associated with protection against reactive nitrogen intermediates, chemotaxis, flagellar and capsule biosynthesis were also upregulated in non-tumour sites of cancer patients. Although they are preliminary, our results further suggest that Fusobacteria may be the leading phylogenetic group responsible for the increase in expression of virulence factors in the oral microbiome of OSCC patients.

References

[1] Ferlay Jacques, Soerjomataram Isabelle, Dikshit Rajesh, Eser Sultan, Mathers Colin, Rebelo Marise, Parkin Donald Maxwell, Forman David, Bray Freddie. Cancer incidence and mortality worldwide: Sources, methods and major patterns in GLOBOCAN 2012. International Journal of Cancer, 2014, 136(5): E359-E386.

[2] Siegel RL, Miller KD, Jemal A. Cancer statistics, 2016. CA Cancer J. Clin., 2016, 66: 7-30.

[3] Gillison ML, Chaturvedi AK, Anderson WF, Fakhry C. Epidemiology of human papillomavirus–positive head and neck squamous cell carcinoma. J. Clin. Oncol., 2015, 33: 3235-3242.

[4] Castellsague X, . HPV involvement in head and neck cancers: comprehensive assessment of biomarkers in 3680 patients. J. Natl. Cancer Inst., 2016, 108: djv403.

[5] Ahn J, Chen CY, Hayes RB. Oral microbiome and oral and gastrointestinal cancer risk. Cancer Causes Control, 2012, 23: 399-404.

[6] Gholizadeh P, . Role of oral microbiome on oral cancers, a review. Biomed. Pharmacother., 2016, 84: 552-558.

[7] Michaud DS, Izard J. Microbiota, oral microbiome, and pancreatic cancer. Cancer J., 2014, 20: 203-206.

[8] Schwabe RF, Jobin C. The microbiome and cancer. Nat. Rev. Cancer, 2013, 13: 800-812.

[9] Zackular JP, . The gut microbiome modulates colon tumorigenesis. mBio, 2013, 4: e00692-e00613.

[10] Bartsch H, Nair J. Chronic inflammation and oxidative stress in the genesis and perpetuation of cancer: role of lipid peroxidation, DNA damage, and repair. Lange. Arch. Surg., 2006, 391: 499-510.

[11] Hardbower DM, de Sablet T, Chaturvedi R, Wilson KT. Chronic inflammation and oxidative stress: the smoking gun for Helicobacter pylori-induced gastric cancer?. Gut Microbes, 2013, 4: 475-481.

[12] Khansari N, Shakiba Y, Mahmoudi M. Chronic inflammation and oxidative stress as a major cause of age- related diseases and cancer. Recent Pat. Inflamm. Allergy Drug Discov., 2009, 3: 73-80.

[13] Kipanyula MJ, . Signaling pathways bridging microbial-triggered inflammation and cancer. Cell. Signal., 2013, 25: 403-416.

[14] Mager DL, . The salivary microbiota as a diagnostic indicator of oral cancer: a descriptive, non-randomized study of cancer-free and oral squamous cell carcinoma subjects. J. Transl. Med., 2005, 3

[15] Hooper SJ, . A molecular analysis of the bacteria present within oral squamous cell carcinoma. J. Med. Microbiol., 2007, 56: 1651-1659.

[16] Pushalkar S, . Comparison of oral microbiota in tumor and non-tumor tissues of patients with oral squamous cell carcinoma. BMC Microbiol., 2012, 12: 144.

[17] Schmidt BL, . Changes in abundance of oral microbiota associated with oral cancer. PLoS ONE, 2014, 9: e98741.

[18] Peterson SN, . Functional expression of dental plaque microbiota. Front. Cell. Infect. Microbiol., 2014, 4: 108.

[19] Simón-Soro A, Guillen-Navarro M, Mira A. Metatranscriptomics reveals overall active bacterial composition in caries lesions. J. Oral Microbiol., 2014, 6: 25443.

[20] Duran-Pinedo AE, . Community-wide transcriptome of the oral microbiome in subjects with and without periodontitis. ISME J., 2014, 8: 1659-1672.

[21] Yost S, Duran-Pinedo AE, Teles R, Krishnan K, Frias-Lopez J. Functional signatures of oral dysbiosis during periodontitis progression revealed by microbial metatranscriptome analysis. Genome Med., 2015, 7

[22] Jorth P, . Metatranscriptomics of the human oral microbiome during health and disease. mBio, 2014, 5: e01012-01014.

[23] Benítez-Páez A, Belda-Ferre P, Simón-Soro A, Mira A. Microbiota diversity and gene expression dynamics in human oral biofilms. BMC Genomics, 2014, 15

[24] Ching T, Huang S, Garmire LX. Power analysis and sample size estimation for RNA-Seq differential expression. RNA, 2014, 20: 1684-1696.

[25] Li K, Bihan M, Yooseph S, Methé BA. Analyses of the microbial diversity across the human microbiome. PLoS ONE, 2012, 7: e32118.

[26] Paster BJ, . Bacterial diversity in human subgingival plaque. J. Bacteriol., 2001, 183: 3770-3783.

[27] Dewhirst FE, . The human oral microbiome. J. Bacteriol., 2010, 192: 5002-5017.

[28] Sussman HH. Iron in cancer. Pathobiology, 1992, 60: 2-9.

[29] Weinberg ED. The role of iron in cancer. Eur. J. Cancer Prev., 1996, 5: 19-36.

[30] Torti SV, Torti FM. Iron and cancer: more ore to be mined. Nat. Rev. Cancer, 2013, 13: 342-355.

[31] Galaris D, Skiada V, Barbouti A. Redox signaling and cancer: the role of “labile” iron. Cancer Lett., 2008, 266: 21-29.

[32] Franco R, Schoneveld O, Georgakilas AG, Panayiotidis MI. Oxidative stress, DNA methylation and carcinogenesis. Cancer Lett., 2008, 266: 6-11.

[33] Murray IA, Patterson AD, Perdew GH. Aryl hydrocarbon receptor ligands in cancer: friend and foe. Nat. Rev. Cancer, 2014, 14: 801-814.

[34] Stanford EA, . Role for the aryl hydrocarbon receptor and diverse ligands in oral squamous cell carcinoma migration and tumorigenesis. Mol. Cancer Res., 2016, 14: 696-706.

[35] Jin L, . Glutamate dehydrogenase 1 signals through antioxidant glutathione peroxidase 1 to regulate redox homeostasis and tumor growth. Cancer Cell, 2015, 27: 257-270.

[36] Liu G, . Glutamate dehydrogenase is a novel prognostic marker and predicts metastases in colorectal cancer patients. J. Transl. Med., 2015, 13

[37] Nokin MJ, . Methylglyoxal, a glycolysis side-product, induces Hsp90 glycation and YAP-mediated tumor growth and metastasis. eLife, 2016, 5: e19375.

[38] Chakraborty S, Gogoi M, Chakravortty D. Lactoylglutathione lyase, a critical enzyme in methylglyoxal detoxification, contributes to survival of Salmonella in the nutrient rich environment. Virulence, 2015, 6: 50-65.

[39] Zhang MM, Ong ClY, Walker MJ, McEwan AG. Defence against methylglyoxal in group A Streptococcus: a role for glyoxylase I in bacterial virulence and survival in neutrophils?. Pathog. Dis., 2016, 74: ftv122.

[40] Chinje EC, Stratford IJ. Role of nitric oxide in growth of solid tumours: a balancing act. Essays Biochem., 1997, 32: 61-72.

[41] Xu W, Liu LZ, Loizidou M, Ahmed M, Charles IG. The role of nitric oxide in cancer. Cell Res., 2002, 12: 311-320.

[42] Perera M, Al-Hebshi NN, Speicher DJ, Perera I, Johnson NW. Emerging role of bacteria in oral carcinogenesis: a review with special reference to perio-pathogenic bacteria. J. Oral Microbiol., 2016, 8: 32762.

[43] Nagy KN, Sonkodi I, Szöke I, Nagy E, Newman HN. The microflora associated with human oral carcinomas. Oral Oncol., 1998, 34: 304-308.

[44] Katz J, Onate MD, Pauley KM, Bhattacharyya I, Cha S. Presence of Porphyromonas gingivalis in gingival squamous cell carcinoma. Int. J. Oral Sci., 2011, 3: 209-215.

[45] Tateda M, . Streptococcus anginosus in head and neck squamous cell carcinoma: implication in carcinogenesis. Int. J. Mol. Med., 2000, 6: 699-703.

[46] Sasaki M, . Streptococcus anginosus infection in oral cancer and its infection route. Oral Dis., 2005, 11: 151-156.

[47] Pushalkar S, . Microbial diversity in saliva of oral squamous cell carcinoma. FEMS Immunol. Med. Microbiol., 2011, 61: 269-277.

[48] Castellarin M, . Fusobacterium nucleatum infection is prevalent in human colorectal carcinoma. Genome Res., 2012, 22: 299-306.

[49] Kostic AD, . Genomic analysis identifies association of Fusobacterium with colorectal carcinoma. Genome Res., 2012, 22: 292-298.

[50] Rubinstein MR, . Fusobacterium nucleatum promotes colorectal carcinogenesis by modulating e-cadherin/β-catenin signaling via its FadA adhesin. Cell Host Microbe, 2013, 14: 195-206.

[51] Duran-Pinedo AE, Paster B, Teles R, Frias-Lopez J. Correlation network analysis applied to complex biofilm communities. PLoS ONE, 2011, 6: e28438.

[52] Gallimidi AB, . Periodontal pathogens Porphyromonas gingivalis and Fusobacterium nucleatum promote tumor progression in an oral-specific chemical carcinogenesis model. Oncotarget, 2015, 6: 22613-22623.

[53] Ahn J, Segers S, Hayes RB. Periodontal disease, Porphyromonas gingivalis serum antibody levels and orodigestive cancer mortality. Carcinogenesis, 2012, 33: 1055-1058.

[54] Inaba H, . Porphyromonas gingivalis promotes invasion of oral squamous cell carcinoma through induction of proMMP9 and its activation. Cell Microbiol., 2014, 16: 131-145.

[55] Sztukowska MN, . Porphyromonas gingivalis initiates a mesenchymal-like transition through ZEB1 in gingival epithelial cells. Cell Microbiol., 2015, 18: 844-858.

[56] Ha NH, . Prolonged and repetitive exposure to Porphyromonas gingivalis increases aggressiveness of oral cancer cells by promoting acquisition of cancer stem cell properties. Tumour Biol., 2015, 36: 9947-9960.

[57] Flynn KJ, Baxter NT, Schloss PD. Metabolic and community synergy of oral bacteria in colorectal cancer. mSphere, 2016, 1: e00102-e00116.

[58] Burns MB, Lynch J, Starr TK, Knights D, Blekhman R. Virulence genes are a signature of the microbiome in the colorectal tumor microenvironment. Genome Med., 2015, 7

[59] Conesa A, . a survey of best practices for RNA-seq data analysis. Genome Biol., 2016, 17

[60] Li H, . The sequence alignment/map format and SAMtools. Bioinformatics, 2009, 25: 2078-2079.

[61] Quinlan AR, Hall IM. BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics, 2010, 26: 841-842.

[62] Davis MPA, van Dongen S, Abreu-Goodger C, Bartonicek N, Enright AJ. Kraken: a set of tools for quality control and analysis of high-throughput sequence data. Methods, 2013, 63: 41-49.

[63] Segata N, . Metagenomic biomarker discovery and explanation. Genome Biol., 2011, 12

[64] Benjamini Y, Hochberg Y. Controlling the false discovery rate: a practical and powerful approach to multiple testing. J. R. Stat. Soc. Ser. B Methodol., 1995, 57: 289-300.

[65] Tarazona S, Garcia-Alcalde F, Dopazo J, Ferrer A, Conesa A. Differential expression in RNA-seq: a matter of depth. Genome Res., 2011, 21: 2213-2223.

[66] Feng J, . GFOLD: a generalized fold change for ranking differentially expressed genes from RNA-seq data. Bioinformatics, 2012, 28: 2782-2788.

[67] Gotz S, . High-throughput functional annotation and data mining with the Blast2GO suite. Nucleic Acids Res., 2008, 36: 3420-3435.

[68] Young MD, Wakefield MJ, Smyth GK, Oshlack A. Gene ontology analysis for RNA-seq: accounting for selection bias. Genome Biol., 2010, 11

[69] Supek F, Bošnjak M, Škunca N, Šmuc T. REVIGO summarizes and visualizes long lists of gene ontology terms. PLoS ONE, 2011, 6: e21800.

[70] Shannon P, . Cytoscape: a software environment for integrated models of biomolecular interaction networks. Genome Res., 2003, 13: 2498-2504.

[71] Sui SJH, Fedynak A, Hsiao WWL, Langille MGI, Brinkman FSL. The association of virulence factors with genomic islands. PLoS ONE, 2009, 4: e8094.

[72] Chen L, Xiong Z, Sun L, Yang J. & Jin, Q. VFDB 2012 update: toward the genetic diversity and molecular evolution of bacterial virulence factors. Nucleic Acids Res., 2012, 40: D641-D645.

[73] Legendre P, Gallagher ED. Ecologically meaningful transformations for ordination of species data. Oecologia, 2001, 129: 271-280.