Immune escape of head and neck cancer mediated by the impaired MHC-I antigen presentation pathway.

Tumor immune evasion is a hallmark of Head and Neck Cancers. The advent of immune checkpoint inhibitors (ICIs) in the first-line setting has transformed the management of these tumors. Unfortunately, the response rate of Head and Neck Squamous Cell Carcinomas (HNSCC) to ICIs is below 15%, regardless of the human papillomavirus (HPV) status, which might be partially related with impaired antigen presentation machinery (APM). Mechanistically, HNSCC cells are usually defective in the expression of MHC-I associated APM, while this transcriptional pathway is critical for the activation of tumor-killing effector T-cells. To specifically illuminate the phenomenon and seek for therapeutic strategies, this review summarizes the most recently identified role of genetic and functional dysregulation of the MHC-I pathway, specifically through changes at the genetic, epigenetic, post-transcriptional, and post-translational levels, which substantially contributes to HNSCC immune escape and ICI resistance. Several treatment modalities can be potentially exploited to restore APM signaling in tumors, which improves anti-tumor immunity through the activation of interferons, vaccines or rimantadine against HPV and the inhibition of EGFR, SHP-2, PI3K and MEK. Additionally, the combinatorial use of radiotherapy or cytotoxic agents with ICIs can synergize to potentiate APM signaling. Future directions would include further dissection of MHC-I related APM signaling in HNSCC and whether reversing this inhibition in combination with ICIs would elicit a more robust immune response leading to improved response rates in HNSCC. Therapeutic approaches to restore the MHC-I antigen presentation machinery in Head and Neck Cancer. (Red color texts represent the according strategies and the outcomes).

Saturated fatty acids dampen the immunogenicity of cancer by suppressing STING.

Oncogenes destabilize STING in epithelial cell-derived cancer cells, such as head and neck squamous cell carcinomas (HNSCCs), to promote immune escape. Despite the abundance of tumor-infiltrating myeloid cells, HNSCC presents notable resistance to STING stimulation. Here, we show how saturated fatty acids in the microenvironment dampen tumor response to STING stimulation. Using single-cell analysis, we found that obesity creates an IFN-I-deprived tumor microenvironment with a massive expansion of suppressive myeloid cell clusters and contraction of effector T cells. Saturated fatty acids, but not unsaturated fatty acids, potently inhibit the STING-IFN-I pathway in HNSCC cells. Myeloid cells from obese mice show dampened responses to STING stimulation and are more suppressive of T cell activation. In agreement, obese hosts exhibited increased tumor burden and lower responsiveness to STING agonist. As a mechanism, saturated fatty acids induce the expression of NLRC3, depletion of which results in a T cell inflamed tumor microenvironment and IFN-I-dependent tumor control.

Treponema denticola Induces Interleukin-36γ Expression in Human Oral Gingival Keratinocytes via the Parallel Activation of NF-κB and Mitogen-Activated Protein Kinase Pathways.

The oral epithelial barrier acts as both a physical barrier to the abundant oral microbiome and a sentry for the immune system that, in health, constrains the accumulation of the polymicrobial plaque biofilm. The immune homeostasis during gingivitis that is largely protective becomes dysregulated, unproductive, and destructive to gingival tissue as periodontal disease progresses to periodontitis. The progression to periodontitis is associated with the dysbiosis of the oral microbiome, with increasing prevalences and abundances of periodontal pathogens such as Treponema denticola. Despite the association of T. denticola with a chronic inflammatory disease, relatively little is known about gingival epithelial cell responses to T. denticola infection. Here, we characterized the transcriptome of gingival keratinocytes following T. denticola challenge and identified interleukin-36γ (IL-36γ) as the most differentially expressed cytokine. IL-36γ expression is regulated by p65 NF-κB and the activation of both the Jun N-terminal protein kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) pathways downstream of Toll-like receptor 2 (TLR2). Finally, we demonstrate for the first time that mitogen- and stress-activated kinase 1 (MSK1) contributes to IL-36γ expression and may link the activation of MAPK and NF-κB signaling. These findings suggest that the interactions of T. denticola with the gingival epithelium lead to elevated IL-36γ expression, which may be a critical inducer and amplifier of gingival inflammation and subsequent alveolar bone loss.

Role of Porphyromonas gingivalis in oral and orodigestive squamous cell carcinoma.

Oral and esophageal squamous cell carcinomas harbor a diverse microbiome that differs compositionally from precancerous and healthy tissues. Though causality is yet to be definitively established, emerging trends implicate periodontal pathogens such as Porphyromonas gingivalis as associated with the cancerous state. Moreover, infection with P. gingivalis correlates with a poor prognosis, and P. gingivalis is oncopathogenic in animal models. Mechanistically, properties of P. gingivalis that have been established in vitro and could promote tumor development include induction of a dysbiotic inflammatory microenvironment, inhibition of apoptosis, increased cell proliferation, enhanced angiogenesis, activation of epithelial-to-mesenchymal transition, and production of carcinogenic metabolites. The microbial community context is also relevant to oncopathogenicity, and consortia of P. gingivalis and Fusobacterium nucleatum are synergistically pathogenic in oral cancer models in vivo. In contrast, oral streptococci, such as Streptococcus gordonii, can antagonize protumorigenic epithelial cell phenotypes induced by P. gingivalis, indicating functionally specialized roles for bacteria in oncogenic communities. Consistent with the notion of the bacterial community constituting the etiologic unit, metatranscriptomic data indicate that functional, rather than compositional, properties of the tumor-associated communities have more relevance to cancer development. A consistent association of P. gingivalis with oral and orodigestive carcinoma could have diagnostic potential for early detection of these conditions that have a high incidence and low survival rates.

Identification and characterization of a UbK family kinase in Porphyromonas gingivalis that phosphorylates the RprY response regulator.

Phosphorylation of proteins is a key component of bacterial signaling systems that can control important functions such as community development and virulence. We report here the identification of a Ubiquitous bacterial Kinase (UbK) family member, designated UbK1, in the anaerobic periodontal pathogen, Porphyromonas gingivalis. UbK1 contains conserved SPT/S, Hanks-type HxDxYR, EW, and Walker A motifs, and a mutation analysis established the Walker A domain and the Hanks-type domain as required for both autophosphorylation and transphosphorylation. UbK1 autophosphorylates on the proximal serine in the SPT/S domain as well as the tyrosine residue within the HxDxYR domain and the tyrosine residue immediately proximal, indicating both serine/threonine and tyrosine specificity. The orphan two-component system response regulator (RR) RprY was phosphorylated on Y41 in the receiver domain by UbK1. The ubk1 gene is essential in P. gingivalis; however, overexpression of UbK1 showed that UbK1-mediated phosphorylation of RprY functions predominantly to augment its properties as a transcriptional enhancer. These results establish that P. gingivalis possesses an active UbK kinase in addition to a previously described Bacterial Tyrosine family kinase. The RR RprY is identified as the first transcriptional regulator controlled by a UbK enzyme.

A bacterial tyrosine phosphatase modulates cell proliferation through targeting RGCC.

Tyrosine phosphatases are often weaponized by bacteria colonizing mucosal barriers to manipulate host cell signal transduction pathways. Porphyromonas gingivalis is a periodontal pathogen and emerging oncopathogen which interferes with gingival epithelial cell proliferation and migration, and induces a partial epithelial mesenchymal transition. P. gingivalis produces two tyrosine phosphatases, and we show here that the low molecular weight tyrosine phosphatase, Ltp1, is secreted within gingival epithelial cells and translocates to the nucleus. An ltp1 mutant of P. gingivalis showed a diminished ability to induce epithelial cell migration and proliferation. Ltp1 was also required for the transcriptional upregulation of Regulator of Growth and Cell Cycle (RGCC), one of the most differentially expressed genes in epithelial cells resulting from P. gingivalis infection. A phosphoarray and siRNA showed that P. gingivalis controlled RGCC expression through Akt, which was activated by phosphorylation on S473. Akt activation is opposed by PTEN, and P. gingivalis decreased the amount of PTEN in epithelial cells. Ectopically expressed Ltp1 bound to PTEN, and reduced phosphorylation of PTEN at Y336 which controls proteasomal degradation. Ltp-1 induced loss of PTEN stability was prevented by chemical inhibition of the proteasome. Knockdown of RGCC suppressed upregulation of Zeb2 and mesenchymal markers by P. gingivalis. RGCC inhibition was also accompanied by a reduction in production of the proinflammatory cytokine IL-6 in response to P. gingivalis. Elevated IL-6 levels can contribute to periodontal destruction, and the ltp1 mutant of P. gingivalis incited less bone loss compared to the parental strain in a murine model of periodontal disease. These results show that P. gingivalis can deliver Ltp1 within gingival epithelial cells, and establish PTEN as the target for Ltp1 phosphatase activity. Disruption of the Akt1/RGCC signaling axis by Ltp1 facilitates P. gingivalis-induced increases in epithelial cell migration, proliferation, EMT and inflammatory cytokine production.

Regulation of olfactomedin 4 by Porphyromonas gingivalis in a community context.

At mucosal barriers, the virulence of microbial communities reflects the outcome of both dysbiotic and eubiotic interactions with the host, with commensal species mitigating or potentiating the action of pathogens. We examined epithelial responses to the oral pathogen Porphyromonas gingivalis as a monoinfection and in association with a community partner, Streptococcus gordonii. RNA-Seq of oral epithelial cells showed that the Notch signaling pathway, including the downstream effector olfactomedin 4 (OLFM4), was differentially regulated by P. gingivalis alone; however, regulation was overridden by S. gordonii. OLFM4 was required for epithelial cell migratory, proliferative and inflammatory responses to P. gingivalis. Activation of Notch signaling was induced through increased expression of the Notch1 receptor and the Jagged1 (Jag1) agonist. In addition, Jag1 was released in response to P. gingivalis, leading to paracrine activation. Following Jag1-Notch1 engagement, the Notch1 extracellular domain was cleaved by P. gingivalis gingipain proteases. Antagonism by S. gordonii involved inhibition of gingipain activity by secreted hydrogen peroxide. The results establish a novel mechanism by which P. gingivalis modulates epithelial cell function which is dependent on community context. These interrelationships have relevance for innate inflammatory responses and epithelial cell fate decisions in oral health and disease.

Role of the RprY response regulator in P. gingivalis community development and virulence.

Porphyromonas gingivalis expresses a limited number of two-component systems, including RprY, an orphan response regulator which lacks a cognate sensor kinase. In this study, we examined cross-phosphorylation of RprY on tyrosine residues and its importance for RprY function. We show that RprY reacts with phosphotyrosine antibodies, and found that the tyrosine (Y) residue at position 41 is predicted to be solvent accessible. Loss of RprY increased the level of heterotypic community development with Streptococcus gordonii, and the community-suppressive function of RprY required Y41. Expression of the Mfa1 fimbrial adhesin was increased in the rprY mutant and in the mutant complemented with rprY containing a Y41F mutation. In a microscale thermophoresis assay, recombinant RprY protein bound to the promoter region of mfa1, and binding was diminished with RprY containing the Y41F substitution. RprY was required for virulence of P. gingivalis in a murine model of alveolar bone loss. Transcriptional profiling indicated that RprY can control the expression of genes encoding the type IX secretion system (T9SS) machinery and virulence factors secreted through the T9SS, including the gingipain proteases and peptidylarginine deiminase (PPAD). Collectively, these results establish the RprY response regulator as a component of the tyrosine phosphorylation regulon in P. gingivalis, which can independently control heterotypic community development through the Mfa1 fimbriae and virulence through the T9SS.

Porphyromonas gingivalis Tyrosine Phosphatase Php1 Promotes Community Development and Pathogenicity.

Protein-tyrosine phosphorylation in bacteria plays a significant role in multiple cellular functions, including those related to community development and virulence. Metal-dependent protein tyrosine phosphatases that belong to the polymerase and histindinol phosphatase (PHP) family are widespread in Gram-positive bacteria. Here, we show that Porphyromonas gingivalis, a Gram-negative periodontal pathogen, expresses a PHP protein, Php1, with divalent metal ion-dependent tyrosine phosphatase activity. Php1 tyrosine phosphatase activity was attenuated by mutation of conserved histidine residues that are important for the coordination of metal ions and by mutation of a conserved arginine residue, a key residue for catalysis in other bacterial PHPs. The php1 gene is located immediately downstream of the gene encoding the bacterial tyrosine (BY) kinase Ptk1, which was a substrate for Php1 in vitro Php1 rapidly caused the conversion of Ptk1 to a state of low tyrosine phosphorylation in the absence of discernible intermediate phosphoforms. Active Php1 was required for P. gingivalis exopolysaccharide production and for community development with the antecedent oral biofilm constituent Streptococcus gordonii under nutrient-depleted conditions. In contrast, the absence of Php1 had no effect on the ability of P. gingivalis to form monospecies biofilms. In vitro, Php1 enzymatic activity was resistant to the effects of the streptococcal secreted metabolites pABA and H2O2, which inhibited Ltp1, an enzyme in the low-molecular-weight (LMW) phosphotyrosine phosphatase family. Ptk1 reciprocally phosphorylated Php1 on tyrosine residues 159 and 161, which independently impacted phosphatase activity. Loss of Php1 rendered P. gingivalis nonvirulent in an animal model of periodontal disease. Collectively, these results demonstrate that P. gingivalis possesses active PHP and LMW tyrosine phosphatases, a unique configuration in Gram-negatives which may allow P. gingivalis to maintain phosphorylation/dephosphorylation homeostasis in multispecies communities. Moreover, Php1 contributes to the pathogenic potential of the organism.IMPORTANCE Periodontal diseases are among the most common infections of humans and are also associated with systemic inflammatory conditions. Colonization and pathogenicity of P. gingivalis are regulated by signal transduction pathways based on protein tyrosine phosphorylation and dephosphorylation. Here, we identify and characterize a novel component of the tyrosine (de)phosphorylation axis: a polymerase and histindinol phosphatase (PHP) family enzyme. This tyrosine phosphatase, designated Php1, was required for P. gingivalis community development with other oral bacteria, and in the absence of Php1 activity P. gingivalis was unable to cause disease in a mouse model of periodontitis. This work provides significant insights into the protein tyrosine (de)phosphorylation network in P. gingivalis, its adaptation to heterotypic communities, and its contribution to colonization and virulence.

Streptococcus gordonii programs epithelial cells to resist ZEB2 induction by Porphyromonas gingivalis.

The polymicrobial microbiome of the oral cavity is a direct precursor of periodontal diseases, and changes in microhabitat or shifts in microbial composition may also be linked to oral squamous cell carcinoma. Dysbiotic oral epithelial responses provoked by individual organisms, and which underlie these diseases, are widely studied. However, organisms may influence community partner species through manipulation of epithelial cell responses, an aspect of the host microbiome interaction that is poorly understood. We report here that Porphyromonas gingivalis, a keystone periodontal pathogen, can up-regulate expression of ZEB2, a transcription factor which controls epithelial-mesenchymal transition and inflammatory responses. ZEB2 regulation by P. gingivalis was mediated through pathways involving ß-catenin and FOXO1. Among the community partners of P. gingivalis, Streptococcus gordonii was capable of antagonizing ZEB2 expression. Mechanistically, S. gordonii suppressed FOXO1 by activating the TAK1-NLK negative regulatory pathway, even in the presence of P. gingivalis Collectively, these results establish S. gordonii as homeostatic commensal, capable of mitigating the activity of a more pathogenic organism through modulation of host signaling.