The vast majority of oral cancer cases are represented by squamous cell carcinoma (SCC) arising at the mucosal surface of the oral cavity. In distinction from oropharyngeal SCC, high-risk Human papillomavirus is rarely associated with oral SCC (OSCC), for which the known major risk factors are tobacco, alcohol, betel quid, especially when used together [1]. Additional risk factors are suspected, because a growing number of OSCC develop in the absence of the known risk factors. Several types of SCC are currently recognized, ranging from those only locally aggressive (verrucous carcinoma) to those capable of metastasis [2]. By far the most common and widely studied is the so-called conventional SCC, a somewhat heterogeneous group ranging in the level of differentiation and aggressiveness. Surgery, irradiation and platinum-based chemotherapy have been the typical treatment modalities for decades with rather little improvement in the impact on patient survival [3]. More recent developments include molecular agents that target important cancer pathways, as well as immunotherapy directed at boosting the adaptive immune response. While newer modalities show promise, particularly when used in combination, the improvements in prognosis so far are small, which necessitates further consideration of other mechanisms.

Another area of study that has the potential to significantly impact upon the approach to patient treatment is the complex host-microbiome interaction network operating throughout the natural history of carcinogenesis in the oral mucosa. Oral mucosa, whether normal or transformed, is colonized largely by commensal organisms, shifting in composition under changing conditions. The resulting interactions may affect both the host and the microbiome, likely adapting to, and influencing, the course of carcinogenesis. So far, no specific microorganism is known to initiate oral carcinogenesis, but evidence does support a role for host-microbial interactions in cancer progression, which is the focus of this review. These interactions will be discussed in the context of normal and transformed surface epithelium.

In this review we attempt to integrate current information on several aspects of oral squamous carcinogenesis, including the colonizing microorganisms, inflammation, and the functions of cell-surface innate pattern recognition receptors that initiate inflammatory and epithelial cell-intrinsic reactions, together capable of promoting carcinogenesis. At first, a discussion of normal structure-function will be presented in order to outline the relationship of the components of interest in the absence of malignant epithelial transformation for comparison with structure-function in carcinogenesis.

The oral cavity is the “lobby” of the digestive tract, a tube subdivided into segments with distinct functions, yet sharing a common goal and some environmental similarities. Under normal conditions, the mucosal surface of the digestive tract begins with stratified squamous epithelium of the oral cavity, pharynx, and esophagus, then changes to glandular epithelium in the stomach, small and large intestines, and back to stratified squamous epithelium at the anorectal junction, reflecting the distinct primary functions of the individual segments. The entire inner surface is continuously exposed to combinations of resident and transient substances and microorganisms, and the epithelial lining, with the help of associated cells and structures, performs barrier function. Areas covered by stratified squamous epithelium deal largely with mechanical forces required to move relatively coarse material and other friction-generating activities (e.g., speech). The tight multilayered structure of the stratified squamous epithelium overlying the basal proliferating cells is well-designed as a strong barrier, especially if the epithelium is keratinizing (forms a stratum corneum). Oral surfaces exposed to more mechanical stress because of mastication, such as gingiva, hard palate, and dorsal surface of the tongue, are covered by keratinizing epithelium, in contrast to non-keratinizing stratified squamous epithelium of the ventral tongue, floor of mouth, buccal, and labial mucosa [4]. The transition to glandular epithelium of the stomach and intestines is necessary for digestion and absorption, while still maintaining a selective barrier.

Besides epithelial cells, immune system dendritic Langerhans cells are normal residents of the stratified squamous epithelium. Additional specialized populations within oral epithelium include melanocytes, Merkel cells, and in some areas of the mouth, taste buds [4]. With the exception of taste buds, specialized populations generally localize in the deep layers, in proximity to the undifferentiated epithelial cells that replenish the rest of the compartment, although Langerhans cells may move up and extend their processes to the epithelial surface. The epithelial cell populations of the stomach and intestines are more variable, and non-epithelial cell types, including mucosa-associated lymphoid tissue (MALT), are found in some portions of the GI tract and the oropharynx. Because MALT is not a feature of the oral mucosa, it is outside the scope of this review. While the structure of the gastrointestinal (GI) tract surfaces is distinct, certain aspects of GI inflammation and carcinogenesis are relevant to the understanding oral carcinogenesis.

The mucosal lamina propria throughout the entire tract is separated from the epithelium by a basement membrane, and is designed to support the local mucosal functions. The typically loose fibrovascular connective tissue of the lamina propria contains both blood and lymphatic vessels, nerve bundles, resident dendritic cells, macrophages, and mast cells, as well as transient leukocyte populations. Salivary gland ducts traverse the lamina propria toward the epithelial surface in gland-bearing oral mucosal sites of the tongue, floor of mouth, labial and buccal mucosa, and posterior palate. Structures deep to the lamina propria differ from one subsite to another.

OSCC develop most often at oral subsites that generally follow geographic variations in population exposures to carcinogens. In the USA, 60% of OSCC occur in the tongue and floor of mouth (sites most exposed to tobacco smoke and alcohol), while in Southeast Asia the most common area is buccal and labial mucosa (typical site for application of the potent carcinogen betel quid), and in Nigeria, 55% of OSCC develop in the gingiva and palate [5–7].

There are several potentially malignant lesions in the oral mucosa [2]. A common early process in oral carcinogenesis that can progress to conventional SCC is the so-called pre-malignant lesion known as epithelial dysplasia (or intraepithelial neoplasia). Early in the process, as the stratified squamous epithelium undergoes transformation, immature atypical keratinocytes originating in the basal layer increasingly occupy more and more of the epithelial compartment, which is usually graded by pathologists as mild, moderate or severe dysplasia, or carcinoma-in-situ—a pre-invasive squamous carcinoma still restricted to the epithelial compartment by the basement membrane [1]. Another, simpler 2-tier system of grading early changes that includes all pre-invasive epithelial transformation is low-grade vs. high-grade dysplasia. Once the transformed epithelial cells breach the basement membrane and move into the lamina propria, the lesion is recognized as invasive squamous cell carcinoma. The loss of epithelial differentiation and maturation leads to structural abnormalities with functional implications. For example, the epithelium can become more permeable due to losses of cellular keratin and disrupted cell-cell adhesion and organization, as demonstrated in studies of skin epidermis [8, 9]. Skin and digestive tract studies indicate that outside-in permeability affects penetration of carcinogens, microbes and microbial products. Inside-out permeability has the potential to impact upon microbial colonization and activity, although the latter has not been tested. The reported changes in oral microbial colonization and virulence (discussed below) may also contribute to epithelial abnormalities and increasing mucosal inflammation. New blood vessels (angiogenesis, vasculogenesis) develop as a consequence of carcinogenesis and inflammation. The abnormal differentiation and uncontrolled growth contribute to disordered OSCC architecture and to the disruption of barrier function, in part through loss of surface continuity in the form of ulcers. SCC are usually ulcerated, which allows microbes and their products to access the connective tissue [10], fueling inflammation. OSCC progress by local invasion into adjacent tissues and metastasize first to regional lymph nodes, then to distant sites. The landscape of the mucosal environment in normal, precancerous, and OSCC conditions is depicted in Figure 1.

Studies of oral microbiome composition and its relationship to OSCC are reviewed extensively elsewhere, so only a brief overview is provided here. Commensal microorganisms, including eubacteria, archaea, protists, fungi, and viruses, inhabit all the epithelial barrier surfaces of the body, where bacteria, in particular, are as numerous as human cells [11, 12]. Oral mucosa is such a surface, and many different types of commensal bacteria appear to constitute the largest group of colonizers [11, 13–15]. Digestive tract segments – oral cavity, esophagus, stomach, small and large intestines—carry different and partially overlapping microbiomes [11], and the intraoral subsites also vary in the amount and diversity of associated organisms. For example, microbial composition of the dorsal tongue with its papillae is much more diverse than that of the buccal and palatal mucosae. In particular, both dorsal tongue and gingival crevices are home to aerobic and anaerobic species that form complex interactive communities, while smooth buccal and palatal mucosae are colonized by aerobes [16]. The normally symbiotic or tolerant relationships between the host and commensals contribute to barrier protection and are important for resistance to colonization by pathogens [17].

Microbiome studies related to oral carcinogenesis report various results. Human observational studies of microbial composition differ because of variations in populations, habits, presence of inflammatory oral disorders, as well as methods of sample collection and analysis [10]. For instance, key OSCC-associated risk factors alone differentially affect the oral microbiome, as revealed by analysis of oral rinses in American adults who are smokers [18] or consumers of alcohol [19]. Investigators also point out that organisms potentially important early in carcinogenesis may no longer be present or enriched in lesions at later stages. A recent study of Indian OSCC patients found distinct microbiomes on surfaces vs. deep intratumoral sites [10], and microorganisms have also been identified in lymph nodes with metastatic OSCC [20].

Although much remains unknown, two oral commensals and periodontal pathogens are under intense interrogation. G-negative anaerobe Fusobacterium (F.) nucleatum in particular has received significant attention in part because of its enrichment at surfaces of early pre-cancerous lesions [21] and at late stages [22] of oral carcinogenesis. One important virulence factor of F. nucleatum is the adhesion molecule FadA which inactivates E-cadherin and causes increased epithelial permeability, as well as facilitates beta-catenin- and WNT-mediated epithelial proliferation, at least demonstrated in colorectal cancer [23, 24]. Porphyromonas (P.) gingivalis is another anaerobic G-negative opportunist in the spotlight. Both F. nucleatum and P. gingivalis are often investigated together for potential roles in oral carcinogenesis because of their contributions to periodontal disease, collaborative growth, an association between periodontal disease and oral cancer [25], and enrichment in the microbiome of both colorectal adenocarcinoma and the pre-cancerous colorectal adenoma [15, 22, 26]. Notably, the majority of epithelial dysplasias do not progress to OSCC, and some subtypes of OSCC develop in the absence of preceding epithelial dysplasia. So far there have been no longitudinal microbiome studies of progressive vs. non-progressive epithelial dysplasia. Other potentially premalignant oral epithelial conditions have not been sufficiently characterized.

Despite study variations, there is overall agreement that the progression from normal to dysplastic squamous epithelium on to OSCC is accompanied by significant shifts in diversity and abundance of microorganisms localized to the lesions, as well as the oral microbiome in general. It is possible that cancer progression and shifting microbiome occur through coevolution, impacted by risk factors and a changing microenvironment.

To test the impact of the oral and tumor-associated microbiomes on OSCC development, several studies have utilized animal models, such as SCC induction by the tobacco-like carcinogen 4-nitroquinolone (4-NQO), or transfer of established human or murine OSCC cells into animals [15]. Similarly, both human and murine oral microbiomes have been evaluated. Overall so far, these studies show that the presence of either selected candidate organisms [26–28] or whole microbiome derived from established tumors, promoted cancer progression in colorectal cancer [29] and OSCC [30]. Even more interesting is that 4-NQO-treated mice colonized by oral microbes from healthy animals developed more and larger OSCC than germ-free 4-NQO-treated mice [30]. In other words, there is evidence that multiple distinct combinations of bacteria identified in either normal conditions or premalignant-malignant lesions are able to enhance the growth of squamous carcinomas initiated by chemical carcinogens. However, a limitation of the reported studies about OSCC growth in germ-free animals is that they have not examined the host immune cell populations and their functions in the associated mucosa. Given that germfree mice are deficient in bone marrow–derived peripheral myeloid populations [31], it is possible that lack of tumor growth in germ-free conditions was at least partly due to the lack of myeloid cells in the tumor microenvironment, disrupting myeloid cell-microbiome-tumor cell interactions.

The inconsistent data about the composition of the OSCC-associated microbiome may be partly balanced by the results of functional analyses of individual species and their combinations. For example, different microorganisms can live in similar conditions and produce similar metabolites [15]. Acetaldehydes, N-nitroso compounds, volatile sulfur compounds, organic acids, and other bacterial metabolites, are capable of causing DNA damage (genotoxicity) and are produced by various organisms found in normal and OSCC-associated microbiomes [15, 32]. Interesting recent advances in metatranscriptomics have revealed that despite variable composition, there was significantly higher microbial metabolic activity and expression of putative virulence factors in established OSCC, with F. nucleatum in the lead, relative to matched sites from tumor-free controls [33]. Increased presence and virulence of certain OSCC-associated bacteria may also be due to the loss of species that otherwise antagonize them [25, 34]. Notably, most of the published reports are about bacteria, in part because many other oral microbiome members have only been discovered recently, and many are not cultivable. The best studied fungal organisms often associated with OSCC are Candida spp. the biology of which is also capable of supporting carcinogenesis [35, 36]. Together, evidence indicates that the microbiomes of established OSCC, regardless of composition, are highly active, and that the products of their metabolism have the potential to directly affect host epithelial cells. It is reasonable to expect that the host-microbiome influences in the evolving OSCC are bidirectional: sharing nutrients, generating and processing metabolites, competing for critical materials and spaces.

A key point of interest for further discussion is that, while the environment of oral epithelial dysplasia and OSCC exhibits shifts in microbial abundance, diversity, and metabolic activities, current understanding is that the colonizers are a mix of commensals and some opportunistic pathogens, all expressing microbe-associated molecular patterns (MAMP) that are common to commensals and pathogens. MAMP are recognized by specific host pattern recognition receptors (PRR) in immune and non-immune cell types, triggering inflammation and other responses. PRR include toll-like receptors (TLR), C-type lectin-like receptors (CLR), and NOD-like receptors (NLR) [37]. Here we will focus on the best studied group, the TLR1-6 expressed on cell surfaces.

TLR are type I transmembrane proteins with critical functions in cells of the immune system. Out of the ten human receptors in this family, the intracellular TLR3, 7, 8, and 9 are localized to acidic organelles called endosomes and recognize microbial nucleic acids, making this group of receptors dependent on phagocytic properties of cells and/or intracellular infections. In contrast, TLR1, 2, 4, 5, and 6 are more accessible and expressed on the plasma cell membranes, and as such detect distinct molecules of microbial surfaces, although TLR4 also associates with endosomes [38]. TLR10 is also expressed on the plasma cell membrane and may be mainly an inhibitory molecule, although the natural ligand for this receptor is unclear [39]. Mice have TLR1-13, the first nine of which are the best studied and are similar to human TLR1-9 in the specificities and functional activities, which is why mice are widely used for TLR-related studies relevant to humans. The cell-surface TLR are of special interest in the context of mucosal function and surface-associated carcinogenesis and will be discussed in more detail.

While exposure to their specific MAMP causes TLR to form homodimers, TLR2 also heterodimerizes with either TLR1 or TLR6, which generates broader recognition of MAMP. TLR1 and TLR6 are not known to function independently of TLR2. TLR2/1 dimers bind bacterial triacylated lipopeptides/lipoproteins, and TLR2/6 dimers interact with diacylated forms of these molecules [40]. Lipoproteins are components of cell walls of G-positive and G-negative bacteria [41]. Both forms of lipoproteins may be expressed in the same species, depending upon the environmental conditions—pH, growth phase, temperature, and salt concentration [40, 42]. Furthermore, peptidoglycan and lipoteichoic acid, thought to interact with TLR2, may not be true TLR2 agonists according to investigations that used more stringent purification methods and other approaches [40]. Molecules and receptors other than TLR2 have been shown to bind peptidoglycans [43]. TLR2/6 also binds fungal cell wall zymosan, a protein-carbohydrate complex. In contrast, TLR4 recognizes the glycolipid lipopolysaccharide (LPS) of most G-negative bacteria. TLR2 and TLR4 are designed to accept transfer of ligands from other host molecules. Molecules that collaborate with TLR2 are CD14, mannose-binding lectin, CD36, and Dectin-1, while TLR4 works with LPS-binding protein (LBP), CD14, and MD2 [38]. The utility of these collaborators is especially evident in responses to whole bacteria. Moreover, several endogenous molecules collectively called danger-associated molecular patterns (DAMP) are reported to also trigger TLR2 or TLR4 activation [44]. Most known DAMP appear to activate TLR indirectly by complexing with other TLR-binding molecules [45]. Of special interest are two DAMP that have TLR-binding and activation abilities, and they are increased during inflammation and cell death, which are abundant in cancer. The high mobility group protein 1 (HMGB1) is fully confirmed to bind TLR4 and trigger its dimerization and signaling without interfering with the LPS-binging site [46], while versican is reported to bind and activate the TLR2/6-CD14 complex [45, 47]. Finally, TLR5 is specific for flagellin, the principal component of flagella in motile organisms, which is often found in pathogens (ex. Salmonella and Helicobacter pylori) and contributes to their virulence [38, 48].

Either commensal or pathogen MAMP binding induces TLR dimerization and signaling. The outcome of signaling depends upon the specific TLR, the cell type, and cell state. Innate immune system monocytes, macrophages and dendritic cells (DC) express high levels of all or most TLR, depending upon cell subset, and have yielded most of the accumulated information about TLR function. TLR signaling is detailed in a recent review by Fitzgerald and Kagan [38] and Vijayan et al. [48]. In brief, ligand-induced activation of all TLR, except TLR3, triggers the assembly of the intracellular multimolecular complex called the myddosome (named after myeloid differentiation primary response protein, or MyD88), which then activates several pathways: canonical NF-kB, mitogen-activated protein kinase (MAPK), and change in metabolism (induction of glycolysis). The endosomal TLR3 activity utilizes the signaling complex called triffosome (named after TIR domain-containing adaptor inducing interferon-β, or TRIF), inducing canonical NF-kB, interferon regulatory factor 3 (IRF3), and other less well-characterized activities. TLR4 is unique in that it uses both the myddosome and the triffosome. Interferon-alpha (IFN-alpha) production is triggered via the triffosome, although in some cells and in response to certain ligands, IFN-alpha may also be induced via the myddosome. The inflammatory pathway signaling in macrophages and DC is further amplified by TLR-induced cytokines that also activate NF-kB (especially IL-1 and TNF-alpha) [38, 48]. Because excessive TLR activity is dangerous to the host [49], there are various levels of cell-intrinsic and extrinsic negative regulation of TLR activities [50, 51]. How negative regulation works in carcinogenesis is under investigation.

Symbiosis with, and tolerance to commensals is the normal state of affairs at surfaces, but this relationship depends upon continuous MAMP recognition via PRR on epithelial and innate immune system cells, the combined efforts of which, in addition to secreted soluble factors (antimicrobial peptides, IgA, other), limit surface penetration by commensal and pathogenic microorganisms [17]. Epithelial MyD88-dependent TLR are required for homeostasis, which was demonstrated in the gut epithelium [52], in epidermal keratinocytes [53] and other cell types, at least in part through induction of cytoprotective factors IL-6, KC-1, and other molecules. Remarkably, signals from the gut microbiome through host PRR have impact well-beyond the gut. These signals control the production, migration and functions of host innate and adaptive immune system cells systemically, which in turn, affects the ability of the host immune system to fight pathogenic infections, as well as local and remote cancer growth and treatment [31, 54–57]. While TLR are expressed by many cell types, the following discussion is focused on immune system cells and epithelial cells because of their critical roles in the mucosa.

In this section, the main focus is on the impact of cell-surface TLR in immune system and epithelial cells on the biology of evolving OSCC with additional information from other surface pre-cancers and carcinomas. There is clear evidence that TLR activity triggered by MAMP or DAMP is highly relevant to the biology of carcinomas because of direct effects on TLR pathways and the consequences that arise from TLR-induced production of soluble factors. Because TLR activity and inflammation are tightly intertwined, they are discussed together. TLR initiate a cascade that begins with activation of canonical (classical) NF-kB and ERK MAPK pathways, amplified by induced host mediators, with subsequent enrollment of STAT3 activities, which affects essentially all cell types present in the tumor. NF-kB and STAT3 activities go hand-in hand in TME. The typical targets of classical NF-kB signaling include factors that affect many aspects of inflammation, cell recruitment, cell proliferation, survival or death, and angiogenesis, such as TNF, IL-1, IL-6, GM-CSF, CXCL8 (IL-8), CXCL1, CXCL2, CCL2, CCL3, CCL5, MMPs, cyclin D1, MYC, BCL-Xl, BCL2, FLIP, COX2, iNOS, VEGF [114], and the hypoxia-inducible factor (HIF)-1, a critical factor in cellular response to hypoxia [131]. On the other hand, the ERK MAPK pathway is responsible for basic cellular processes, including cell proliferation and differentiation, which are crucial for cancer cells, including cell proliferation, survival, growth, metabolism, migration, and differentiation [132]. The MAPK pathway is important for cell-intrinsic effects of TLR signaling.

The comprehensive approach to this review was used in order to develop a more complete landscape of oral carcinogenesis in the context of microbial colonization and cell-surface PRR designed to perform many functions to protect the host from infections and dangerous runaway immune reactions. The evidence discussed here indicates that in the slow process of carcinogenesis, the initially destructive properties of cell-surface TLR activation are tightly controlled and usually short-lived, and anti-destructive mechanisms take over, in a way consistent with the process in infections or injury. The overwhelming input of many DAMP- and MAMP-mediated signals into malignant cells, stromal immune cells and non-immune cells, works against the few available toxic mechanisms that are necessary to destroy the malignant cells, and complicates the search for effective treatments. A more comprehensive approach to patient treatment throughout the process of carcinogenesis that incorporates better understanding of TLR contributions to the evolving or established TME has the potential to improve patient outcomes.

ZK: conceptual design, acquisition and analysis of relevant publications, outline for potential figures, and writing of the manuscript. JL: acquisition and analysis of relevant literature, design and production of figures, and writing portions of the manuscript. All authors contributed to the article and approved the submitted version.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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