The Microbiome of Oral Squamous Cell Carcinomas: a Functional Perspective

The vast majority of microbes designated as Class 1 carcinogens by the International Agency for Research on Cancer (IARC) are viruses. Therefore, viruses come to mind first when raising a question regarding the role of microorganisms in cancer. As far as HNCs are concerned, two families of oncogenic viruses are particularly important: Human Papillomaviruses (HPVs) and Human Herpesviruses (HHVs). There is currently incontrovertible evidence that a small number of so-called high-risk (hr) HPVs are responsible for a global epidemic of oropharyngeal cancer. By now, cancer of the oropharynx has replaced cancer of the uterine cervix as the most common HPV-related cancers in the USA [20]. A smaller proportion of intra-oral cancers are also caused by hrHPVs. Although these HPVs are epitheliotropic, cancerous lesions mostly arise in the mucosa associated with lymphoid tissue, due to interactions between lymphocytes and keratinocytes, namely in the posterior third of the tongue (such lesions located in the palatine tonsils and elsewhere in Waldeyer’s tonsillar ring are not classified as intra-oral). The underlying cellular mechanisms are well understood: HPV E6 and HPV E7 oncogenes interfere with p53 and retinoblastoma gene proteins, respectively, and thereby block their tumor suppressor actions [21]. Consequently, there are encouraging possibilities to block the oncogenic pathways with interfering ribonucleic acid (RNA) or by gene editing [22].

Among the HHVs, the Epstein-Barr virus (HHV-4) is the major cause of nasopharyngeal cancers, a distinct biological entity [23]. However, there is limited evidence that HHV-4 may play a role in oral cancer [21, 24]. HHV-8 is the etiologic agent for Kaposi’s sarcoma located in the mouth and elsewhere that is prevalent in immunosuppressed individuals, particularly in patients with acquired immune deficiency syndrome (AIDS) [21, 25]. The virus is carried in the oropharynx and can be recovered from saliva/oral fluid samples [21, 26]. There is circumstantial evidence that Herpes Simplex Viruses (HSV), both HSV-1 and HSV-2, are associated with oral (and cervical) squamous cell carcinoma in humans, as well as supportive animal studies [21], but a direct oncogenic role remains unsubstantiated [26].

Apart from human viruses, the oral cavity is also home to a complex community of bacteriophages that are thought to influence the ecology and pathogenicity of the oral bacterial community [27]. Whether or not this phage community plays a role in oral carcinogenesis has not been explored.

In the 1990s, the relationship between bacteria and carcinogenesis was first established by demonstrating the causative role of Helicobacter pylori in gastric cancer [28], and since then, tremendous efforts have been invested in exploring the relationship between bacteria and cancer in sites elsewhere in the body. This has led to uncovering additional associations, such as that of Chlamydia trachomatis with cervical cancer [29], Salmonella typhi with gallbladder cancer [30], and Bacteroides fragilis with colon cancer [31]. As far as oral cancer is concerned, evidence is emerging, primarily from in vitro and animal studies, for the carcinogenicity of the periodontal bacteria Porphyromonas gingivalis (P. gingivalis) and Fusobacterium nucleatum (F. nucleatum). Interest in these two bacteria from the oral microbiome as potential carcinogens has been fueled by studies that implicated them in pancreatic and colorectal cancers (CRC), respectively [32‐35]. The mechanisms by which they are thought to contribute to oral carcinogenesis are shown conceptually in Fig. 2 [11].

P. gingivalis has been shown to inhibit apoptosis at different levels, including activation of JAK1/STAT3 and PI3K/Akt signaling pathways [36, 37], suppression of proapoptotic BCL-2-associated death promoter [38], blocking activity of caspase-3 and caspase-9 [37, 38], upregulation of microRNA-203 [39], and prevention of ATP-dependent P2X₇-mediated apoptosis [40]. Both P. gingivalis and F. nucleatum activate cell proliferation through upregulation of kinases and cyclins [41‐43], activation of the β-catenin signaling pathway [44, 45], and downregulating level of the p53 tumor suppressor [41]. They also have been found to enhance cellular invasion, primarily through upregulation/activation of matrix metalloproteinases, including MMP-1, MMP-9, MMP-10, and MMP-13, and inducing stemness and epithelial to mesenchymal transition (EMT) [43, 46‐48]. In addition, P. gingivalis and F. nucleatum are believed to contribute to progression of oral cancer by inducing chronic inflammation via increasing production of pro-inflammatory cytokines [49‐52]. Production of carcinogenic substances, such as acetaldehyde (from ethanol), may also play an important role, but has not been documented for these two species. Nonetheless, there is evidence for such production by other oral microorganism species, such as Streptococci [53], Neisseria [54], and Candida [55, 56].

Despite the convincing evidence from in vitro studies on the carcinogenicity of P. gingivalis and F. nucleatum just summarized, evidence for a direct carcinogenic role in oral cancer based on clinical studies is still lacking, as described in the following section.

Clinical studies that assessed the association between bacteria and cancers of the oral cavity are summarized in Table 1. Most of these studies restricted inclusion to OSCC samples, but a number of them also included HNCs located at sites other than the oral cavity (parts of the pharynx, larynx, or esophagus) as well as lesions that were only potentially malignant. Only two decades ago, namely in 1998, Nagy and collaborators first demonstrated differences in composition of the microbial community colonizing the surface of OSCC and adjacent normal tissue, using culture techniques [57•]. Between 2000 and 2005, Streptococcus anginosus (S. anginosus) became implicated in the etiology of HNC, including OSCC, by research groups from Japan [58, 59, 61]. However, these studies suffered from lack of proper control samples: the high detection rates of the bacterium observed in HNC samples were simply because it is a normal colonizer of the oropharynx. Interest in S. anginosus thus faded quickly.

In 2006, Hooper et al. demonstrated, for the first time, the presence of a viable complex bacterial community within the OSCC tissues that is compositionally different from that found in the tumors’ healthy margins [62•]. Since then, the vast majority of investigations have employed sequencing of the 16S rRNA (ribosomal RNA) gene to characterize the microbiomes in study samples, initially with the Sanger method and more recently with NGS chemistries [63, 65‐70, 71•, 72‐79, 80•, 81, 82, 83•, 84]. Nevertheless, there are significant methodological differences between these studies in terms of (1) the type of samples analyzed (saliva (stimulated or unstimulated), surface swabs, or biopsies); (2) the nature of control or comparison samples used (tumor-adjacent, clinically normal; contralateral to tumor; healthy subject; or benign lesions); (3) the hypervariable region of the 16S rRNA gene selected for sequencing (e.g., V1–V3 or V4–V5); and (4) the bioinformatic analysis method used for analysis of sequencing data (sequence quality control, reference database, and taxonomy assignment algorithm). The latter particularly affects taxonomic resolution, with only a few studies reporting species-level profiles and the rest limited to the genus level.

As displayed in Table 1, a number of bacterial taxa, particularly Fusobacterium spp., and to a lesser extent, Campylobacter, Parvimonas, and Prevotella spp., were repeatedly found to be significantly enriched in OSCC samples (Table 1), whereas Streptococci frequently were found in association with health. Nevertheless, a more careful examination of the results from these studies reveals there are significant variations (and sometimes contradictions) in the composition of the bacteriome associated with tumors from one cohort to another, and even from one subject to another within the same cohort, especially at the species level. Overall, there is, therefore, not sufficient evidence to implicate specific bacterial species or any consortium thereof in the etiology of OSCC. Interestingly, only one of these studies identified P. gingivalis in association with oral cancer [64].

While the observed variation in composition of the bacteriome associated with oral cancer may, at least in part, be attributed to the methodological differences among studies described above, it may also be explained by the fact that different species frequently can serve the same functions in their communities and thus substitute for each other, a phenomenon called functional redundancy [87••]. That is, a microbial community with a certain subset of species enriched may perform the same function as another community with a different subset of overabundant species. This concept can be likened to player substitution in team sports like soccer. Indeed, functional analyses of bacteriomes associated with oral cancer produced more consistent results than those obtained with compositional profiling reflecting only the abundance/presence of specific individual members of the bacteriome. Some of the studies listed in Table 1 have used Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt) to infer metagenomes from the 16S rRNA profiles obtained (functional prediction) [71•, 79, 82, 83•]. Furthermore, in 2018 and for the first time, Yost and team used metatranscriptome sequencing to explore the transcriptional activity (gene expression) of the microbiome associated with OSCC [85••]. In these studies, the tumor-associated bacteriomes possessed similar functional signatures despite variation in their compositional profiles. For example, enrichment of primarily pro-inflammatory features, such as LPS biosynthesis, flagella assembly, bacterial chemotaxis, and production of peptidases, were enriched in the tumors, while activities like glycolysis and gluconeogenesis, amino acid synthesis and metabolism, and DNA repair were enhanced in the healthy samples. The study by Yost and colleagues identified additional virulence factors associated with OSCC, including increased expression of tryptophanase, superoxide dismutase, hemolysins, and adhesins [85••]. More details about the microbiome functional features associated with OSCC identified in this study are shown in Fig. 3 [85••].