“Messieurs, c'est les microbes qui auront le dernier mot”: Gentlemen, it is the microbes who have the last word (Louis Pasteur)—Fusobacterium nucleatum protect tumors from killing by immune cells

Abstract

Fusobacterium nucleatum is present in colon cancers where it was shown to generate a proinflammatory microenvironment that supports colorectal neoplasia progression. Remarkably, alongside with proinflammatory stimulation, fusobacteria also inhibit cytotoxicity of immune cells. Thus, it appears as if tumors exploit fusobacteria to generate a favorable proinflammatory and anti-cytotoxic microenvironment.

Keywords:

• tumor immunity
• NK cells
• fusobacteria
• colon adenocarcinoma

Viruses were the first infectious agents shown able to induce cancer. Over a century ago, in 1911, Rous demonstrated that sarcoma can be induced in chickens by a virus.¹ Fifty-five years later (1966) the association of viruses with human cancer was reported by Epstein, Achong and Barr who used electron microscopy to demonstrate the existence of Epstein-Barr Virus in Burkitt lymphoma cells.² Following studies linked hepatitis B and C viruses with liver cancer, papillomavirus with cervical cancer and herpesviruses with Kaposi sarcoma. Currently about 20% of cancer incidence are linked to infectious agents.³

In contrast to the rising understanding of the oncogenic potential of viruses, no substantial evidence linking bacteria with cancer progression were available until the end of the twentieth century. In fact, bacteria were used as agents for anticancer treatment. In the early 1890s, a surgeon named William Coley pioneered the use of bacteria (mainly Streptococcus pyogenes) and their extracts to evoke antitumor immunity and successfully treat cancer patients. Later, radiotherapy, chemotherapy and immunotherapy became the main strategies for cancer treatment.

The realization that Helicobacter pylori is a causative agent of gastric cancers in the 1990s indicated, for the first time, that bacteria is involved in tumor promotion. Indeed, it was shown recently that mice that are genetically susceptible to colorectal cancer (CRC) develop significantly fewer tumors under germ-free conditions than when they have a conventional microbiota.⁴ Among the potential bacterial cancer drivers are Escherichia coli strains that generate a mutagenic toxin called colibactin that can induce single-strand DNA breaks; and fragilysin expressing Bacteroides fragilis that is both genotoxic and capable of cleavage of the tumor suppressor protein E-cadherin.⁴ A recent and surprising bacterial species added to cancer associated ones is F. nucleatum.

F. nucleatum is a common oral non-spore forming gram-negative anaerobe. It is also involved in extraoral infections including preterm births. Recent genomics and transcriptomics evidence demonstrated that F. nucleatum is also prevalent in colorectal carcinoma.⁴

Tumors are monitored and controlled by the immune system. Natural Killer (NK) cells are large granular lymphocytes that comprise ∼10% of peripheral blood lymphocytes. They are best known for their ability to kill transformed and virally infected cells and for secreting cytokines, specifically TNF-α and IFNγ. NK cell activity is regulated through a balance of signals derived from inhibitory and activating receptors. Direct recognition of bacteria by NK cells was also demonstrated and interestingly bacterial pathogens have adapted and evolved mechanisms to escape or manipulate the NK cell recognition. For example, NK cells control urinary pathogenic E. coli (UPEC) in the urinary tract. However, UPEC was found to escape this control by type1 fimbriae mediated attachment to NK cells followed with a rapid killing of the NK cells via the hemolysinA toxin.⁵ Whether adherent-invasive E. coli kills NK cells similar to uropathogenic E. coli in order to promote colitis-associated CRC is yet to be determined.

In a mouse model of periodontal disease NK cells were shown to specifically recognize an unknown proteinous ligand on F. nucleatum via their killer receptor NCR1 (NKp46 in humans). This recognition is followed by TNF-α secretion from the NK cells and results in the aggravation of periodontal disease in mice challenged with fusobacteria.⁶ Since NK cells play a major role in tumor control and fusobacteria are abundant in colorectal adenocarcinoma, it was rational to test the effect of fusobacteria on the ability of NK cells to kill tumors.

Surprisingly, killing of various tumors by NK cells was inhibited by the presence of various F. nucleatum strains. Inhibition of tumor-killing by fusobacteria was found to be mediated by human, but not by mouse TIGIT, an inhibitory receptor present on all human NK cells and on various T cells. Using a library of F. nucleatum transposon-inserted mutants, the Fap2 lectin of F. nucleatum previously associated with lymphocyte apoptosis by fusobacteria,⁷ was found to directly interact with TIGIT. This TIGIT activation by fusobacteria leads to the inhibition of NK cell cytotoxicity. TIGIT inhibition was dependent on the hemagglutination ability of the bacterium; strains able to cause hemagglutination via Fap2 activate TIGIT, whereas strains unable to cause hemagglutination do not activate TIGIT. TIGIT is expressed on a variety of T cells including tumor-infiltrating lymphocytes (TILs). The TIGIT expressing T cells were also inhibited by F. nucleatum via Fap2 (Fig. 1).⁸ Thus, a bacterium-dependent, tumor immune evasion mechanism in which tumors exploit the Fap2 protein of F. nucleatum to inhibit immune cell activity via TIGIT was revealed.

Figure 1. Fusobacterium nucleatum inhibits immune cell activity through the interaction with TIGIT. Tumor-infiltrating lymphocytes (TILs, including NK cells) are recruited to tumors, activated and commence in tumor regression. This immune response results in tumor regression (top). The Fap2 lectin expressed by fusobacteria that attach to tumors, activate TIGIT suppressive ability. TIGIT activation by fusobacteria leads to the inhibition of the TIL's cytotoxicity thus enabling tumor growth (bottom panel).

Prospective

Whether fusobacteria initiate CRC or colonize preexisting tumors remains to be determined. So far, evidence suggests that fusobacteria colonize adenomas or pre-existing tumors rather than driving tumor initiation.^8-10 It will be interesting to investigate whether other tumor promoting bacteria interact with TIGIT. It also remains to be determined whether blocking the ability of fusobacteria to activate TIGIT will improve tumor control by the host's immunity. Finally, since F. nucleatum efficiently penetrates colon cancer, perhaps it can be armed with an anticancer drug and used as a novel modality for anti-CRC treatment.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.