Fibroblasts from HPV-negative oropharynx squamous cell carcinomas stimulate the release of osteopontin from cancer cells via the release of IL-6.

Introduction: HPV-associated oropharyngeal squamous cell carcinoma (OPSCC) shows distinct biological and clinical behaviour when compared to HPV-negative OPSCC. The overall role of the tumour microenvironment (TME) in head and neck cancer progression and metastasis has been studied intensively, but differences in HPV-negative and HPV-positive OPSCCs are less understood. Objective: To investigate the role of cancer-associated fibroblasts (CAFs) and the functional interactions of normal tonsil fibroblasts (NTFs) and OP CAFs with HPV+ and HPV- OPSCC cells and explore novel candidates in tumour-fibroblast crosstalk. Materials and methods: A retrospective cohort of 143 primary OPSCCs was characterised using HPV16/18 RNAScope assay, p16 IHC and ɑ-SMA. Four OPSCC, three NTF and 2 new OPSCC CAF cultures were used to assess the cytokine-based interactions using cytokine arrays on conditioned media (CM), followed by co-culture approaches to identify the role of individual cell types and the role of OPN (SPP1) and IL-6 in SCC/fibroblast communication. Results: HPV status was associated with better overall survival. Although ɑ-SMA expression was observed in both OPSCC subtypes, it provided survival stratification only in the HPV-positive group (Log-Rank p = 0.02). Three normal tonsillar fibroblast cultures (NTFs) were characterised by induction of myofibroblastic and senescent phenotypes with similar reactivity to our published NOF phenotype. The OPSCC-derived CAF cultures were characterised and their baseline myofibroblastic and senescence phenotypes varied. Cytokine array analysis of CM to identify novel candidates in the crosstalk between OPSCC tumour cells and NTFs/CAFs identified differences in the cytokine profiles on comparison of HPV+ and HPV- OPSCC cells. Osteopontin (OPN/SPP1) was identified, particularly in HPV-negative OPSCC cell analyses. We have demonstrated that OPN was produced by the OPSCC cells and revealed an associated upregulation of IL-6 in fibroblasts. Treatment of NTFs with rOPN showed alteration in phenotype, including increased contraction and IL-6 production. Antibody-mediated inhibition of CD44v6 attenuated the production of IL-6 by OPN in NTFs. Conclusion: This investigation with OPSCC fibroblasts provides novel insights into the role of CAFs in OPSCC mediated by IL-6 stimulated release of OPN from HPV negative OPSCC cells. The details of HPV-positive SCC cell/fibroblast cytokine crosstalk remain elusive.

A Cancer Cell-Intrinsic GOT2-PPARδ Axis Suppresses Antitumor Immunity.

Despite significant recent advances in precision medicine, pancreatic ductal adenocarcinoma (PDAC) remains near uniformly lethal. Although immune-modulatory therapies hold promise to meaningfully improve outcomes for patients with PDAC, the development of such therapies requires an improved understanding of the immune evasion mechanisms that characterize the PDAC microenvironment. Here, we show that cancer cell-intrinsic glutamic-oxaloacetic transaminase 2 (GOT2) shapes the immune microenvironment to suppress antitumor immunity. Mechanistically, we find that GOT2 functions beyond its established role in the malate-aspartate shuttle and promotes the transcriptional activity of nuclear receptor peroxisome proliferator-activated receptor delta (PPARδ), facilitated by direct fatty acid binding. Although GOT2 is dispensable for cancer cell proliferation in vivo, the GOT2-PPARδ axis promotes spatial restriction of both CD4+ and CD8+ T cells from the tumor microenvironment. Our results demonstrate a noncanonical function for an established mitochondrial enzyme in transcriptional regulation of immune evasion, which may be exploitable to promote a productive antitumor immune response. SIGNIFICANCE: Prior studies demonstrate the important moonlighting functions of metabolic enzymes in cancer. We find that the mitochondrial transaminase GOT2 binds directly to fatty acid ligands that regulate the nuclear receptor PPARδ, and this functional interaction critically regulates the immune microenvironment of pancreatic cancer to promote tumor progression. See related commentary by Nwosu and di Magliano, p. 2237.. This article is highlighted in the In This Issue feature, p. 2221.

SMN-deficient cells exhibit increased ribosomal DNA damage.

Spinal muscular atrophy, the leading genetic cause of infant mortality, is a motor neuron disease caused by low levels of survival motor neuron (SMN) protein. SMN is a multifunctional protein that is implicated in numerous cytoplasmic and nuclear processes. Recently, increasing attention is being paid to the role of SMN in the maintenance of DNA integrity. DNA damage and genome instability have been linked to a range of neurodegenerative diseases. The ribosomal DNA (rDNA) represents a particularly unstable locus undergoing frequent breakage. Instability in rDNA has been associated with cancer, premature ageing syndromes, and a number of neurodegenerative disorders. Here, we report that SMN-deficient cells exhibit increased rDNA damage leading to impaired ribosomal RNA synthesis and translation. We also unravel an interaction between SMN and RNA polymerase I. Moreover, we uncover an spinal muscular atrophy motor neuron-specific deficiency of DDX21 protein, which is required for resolving R-loops in the nucleolus. Taken together, our findings suggest a new role of SMN in rDNA integrity.

Mesenchymal Lineage Heterogeneity Underlies Nonredundant Functions of Pancreatic Cancer-Associated Fibroblasts.

Cancer-associated fibroblast (CAF) heterogeneity is increasingly appreciated, but the origins and functions of distinct CAF subtypes remain poorly understood. The abundant and transcriptionally diverse CAF population in pancreatic ductal adenocarcinoma (PDAC) is thought to arise from a common cell of origin, pancreatic stellate cells (PSC), with diversification resulting from cytokine and growth factor gradients within the tumor microenvironment. Here we analyzed the differentiation and function of PSCs during tumor progression in vivo. Contrary to expectations, we found that PSCs give rise to a numerically minor subset of PDAC CAFs. Targeted ablation of PSC-derived CAFs within their host tissue revealed nonredundant functions for this defined CAF population in shaping the PDAC microenvironment, including production of specific extracellular matrix components and tissue stiffness regulation. Together, these findings link stromal evolution from distinct cells of origin to transcriptional heterogeneity among PDAC CAFs and demonstrate unique functions for CAFs of a defined cellular origin. SIGNIFICANCE: By tracking and ablating a specific CAF population, we find that a numerically minor CAF subtype from a defined cell of origin plays unique roles in establishing the pancreatic tumor microenvironment. Together with prior studies, this work suggests that mesenchymal lineage heterogeneity and signaling gradients diversify PDAC CAFs.See related commentary by Cukierman, p. 296.This article is highlighted in the In This Issue feature, p. 275.

RARß Expression in Keratinocytes from Potentially Malignant Oral Lesions: The Functional Consequences of Re-Expression by De-Methylating Agents.

Loss of RARß2 expression by promoter methylation is an early event in oral carcinogenesis. Understanding the mechanisms and consequences of RARß loss may aid in understanding the disappointing results of retinoid chemoprevention trials. This study aimed to describe the effects of all-trans retinoic acid (ATRA) and the de-methylating agent 5-Aza-2' deoxycytidine (5-AZA-CdR) on a panel of immortal potentially malignant oral lesion (PMOL) cell cultures. RARß expression was assessed in PMOL tissues by immunohistochemistry. Cells were treated with ATRA ± 5-AZA-CdR, and the effects on the cell cycle and senescence were assessed. In PMOL tissues, RARß expression was variable, but lower in biopsies which gave rise to immortal cell cultures. Treatment of iPMOL cells with ATRA resulted in little change in RARß expression, but the addition of 5-AZA-CdR resulted in significant increases. The effects on the cell cycle and senescence were variable and may be related to 5-AZA-CdR, as this has wider effects on the cell cycle. Overall, the response of iPMOL cells to ATRA and 5-AZA-CdR treatment was variable and is dependent on several factors, including RARß-promoter methylation. These findings may help to explain the lack of consistent effect of retinoids in PMOLs seen in chemoprevention trials.

The role of icIL-1RA in keratinocyte senescence and development of the senescence-associated secretory phenotype.

There is compelling evidence that senescent cells, through the senescence-associated secretory phenotype (SASP), can promote malignant transformation and invasion. Interleukin-1 (IL-1) is a key mediator of this cytokine network, but the control of its activity in the senescence programme has not been elucidated. IL-1 signalling is regulated by IL-1RA, which has four variants. Here, we show that expression of intracellular IL-1RA type 1 (icIL-1RA1), which competitively inhibits binding of IL-1 to its receptor, is progressively lost during oral carcinogenesis ex vivo and that the pattern of expression is associated with keratinocyte replicative fate in vitro We demonstrate that icIL-1RA1 is an important regulator of the SASP in mortal cells, as CRISPR/Cas9-mediated icIL-1RA1 knockdown in normal and mortal dysplastic oral keratinocytes is followed by increased IL-6 and IL-8 secretion, and rapid senescence following release from RhoA-activated kinase inhibition. Thus, we suggest that downregulation of icIL-1RA1 in early stages of the carcinogenesis process can enable the development of a premature and deregulated SASP, creating a pro-inflammatory state in which cancer is more likely to arise.

Glandular Odontogenic Cyst with Metaplastic Cartilage: Report of an Unusual Case and Literature Review.

Glandular odontogenic cysts are rare odontogenic cysts with a wide range of histopathological features. In this paper we describe the clinical and pathological features of an unusual case of a glandular odontogenic cyst with metaplastic cartilage. The previous literature of odontogenic cysts presenting with metaplastic cartilage is reviewed alongside a discussion of the differential diagnoses. To our knowledge this is the first reported case of a glandular odontogenic cyst with metaplastic cartilage.

Multiple Orthokeratinized Odontogenic Cysts: A Report of Two Cases and Review of the Literature.

Orthokeratinized odontogenic cysts (OOC) are developmental odontogenic cysts characterised by an orthokeratinized stratified squamous epithelial lining. They were originally believed to be part of the spectrum of Odontogenic Keratocyst, but are now considered to be a distinct entity. They are rare, making up approximately 1% of all odontogenic cysts and they usually occur singly. In this paper we present two new cases of multiple OOCs, and compare them to previous case reports of multiple lesions. The clinical and pathological features are discussed, along with possible diagnostic pitfalls.

Fibroblasts as Modulators of Local and Systemic Cancer Metabolism.

Fibroblast activation is an accompanying feature of solid tumor progression, resembling a conserved host response to tissue damage. Cancer-associated fibroblasts (CAFs) comprise a heterogeneous and plastic population with increasingly appreciated roles in tumor growth, metastatic capacity, and response to therapy. Classical features of fibroblasts in a wound-healing response, including profound extracellular matrix production and cytokine release, are recapitulated in cancer. Emerging evidence suggests that fibroblastic cells in the microenvironments of solid tumors also critically modulate cellular metabolism in the neoplastic compartment through mechanisms including paracrine transfer of metabolites or non-cell-autonomous regulation of metabolic signaling pathways. These metabolic functions may represent common mechanisms by which fibroblasts stimulate growth of the regenerating epithelium during a wound-healing reaction, or may reflect unique co-evolution of cancer cells and surrounding stroma within the tumor microenvironment. Here we review the recent literature supporting an important role for CAFs in regulation of cancer cell metabolism, and relevant pathways that may serve as targets for therapeutic intervention.

Lumps and Bumps of the Gingiva: A Pathological Miscellany.

Lesions of the gingivae are amongst the commonest lesions seen in patients and the vast majority are reactive hyperplasias, related to a number of chronic irritant stimuli. However, there are a number of entities that have a predilection for the gingivae, which are much less common in other parts of the oral cavity. The purpose of this paper is to discuss the clinical and histological differential diagnoses when presented with a lump on the gingivae, including the approach to diagnosis and diagnostic pitfalls.

Structure-Activity Relationships of Central Nervous System Penetration by Fatty Acid Amide Hydrolase (FAAH)-Targeted Thyromimetic Prodrugs.

Thyroid hormone (TH) action is of clinical interest in treating demyelinating diseases of the central nervous system (CNS). Two amide prodrugs of sobetirome, a potent thyroid hormone agonist, were previously shown to significantly improve CNS selective distribution of the parent drug through hydrolysis in the CNS by fatty acid amide hydrolase (FAAH). This concept is elaborated upon here with a series of 29 amide prodrugs targeting FAAH. We identify that conservative aliphatic modifications such as the N-methyl (4), N-ethyl (5), N-fluoroethyl (15), and N-cyclopropyl (18) substantially favor selective CNS distribution of the parent drug in mice. Additionally, lead compounds exhibit moderate to good rates of hydrolysis at FAAH in vitro suggesting both enzymatic and physicochemical properties are important parameters for optimization. Both 4 and 15 were orally bioavailable while retaining appreciable CNS parent drug delivery following an oral dose. The pharmacokinetic parameters of 4 over 24 h postdose (i.v. and p.o.) were determined.

Odontogenic tumors and lesions treated in a single specialist oral and maxillofacial pathology unit in the United Kingdom in 1992-2016.

OBJECTIVES: Odontogenic tumors (ODTs) are a heterogeneous group of lesions derived from elements of tooth-forming tissues. No detailed data on the incidence of odontogenic tumors in the United Kingdom have been published. The aim of this study was to retrospectively describe the range and incidence of odontogenic tumors from 1992 to 2016 in a single specialist unit and to compare this population with others. STUDY DESIGN: By using the Oral and Maxillofacial Pathology database, Sheffield (UK), we included both local and referred consultation cases. A proportion of diagnoses were reclassified in accordance with the 2017 World Health Organization classification. RESULTS: In total, 559 odontogenic tumors were diagnosed. Overall, the most common lesions were ameloblastoma (196 [33.8%]), odontoma (148 [25.5%]), and odontogenic myxoma (37 [6.3%]), but this varied between local and referral case populations, with odontomas being most common in the local population (43%). The sites affected and the gender and age of patients were similar to other Western populations. Malignant ODTs comprised 33 cases (5.7%), of which 9 (27.3%) were ameloblastic carcinoma. The majority of the malignant ODTs comprised referral cases. CONCLUSIONS: Here, we present the first detailed data on ODTs within a UK population, and the pattern of incidence from the local population is similar to other Western populations. The exceptional rarity of malignant ODTs emphasizes the need for specialist centers for their treatment to gain diagnostic experience.

Targeting Fatty-Acid Amide Hydrolase with Prodrugs for CNS-Selective Therapy.

The blood-brain barrier (BBB) can be a substantial impediment to achieving therapeutic levels of drugs in the CNS. Certain chemical functionality such as the carboxylic acid is a general liability for BBB permeability preventing significant CNS distribution of a drug from a systemic dose. Here, we report a strategy for CNS-selective distribution of the carboxylic acid containing thyromimetic sobetirome using prodrugs targeted to fatty-acid amide hydrolase (FAAH), which is expressed in the brain. Two amide prodrugs of sobetirome were shown to be efficient substrates of FAAH with Vmax/KM values comparable to the natural endocannabinoid FAAH substrate anandamide. In mice, a systemic dose of sobetirome prodrug leads to a substantial ∼60-fold increase in brain distribution (Kp) of sobetirome compared to an equimolar systemic dose of the parent drug. The increased delivery of sobetirome to the brain from the prodrug was diminished by both pharmacological inhibition and genetic deletion of FAAH in vivo. The increased brain exposure of sobetirome arising from the prodrug corresponds to ∼30-fold increased potency in brain target engagement compared to the parent drug. These results suggest that FAAH-targeted prodrugs can considerably increase drug exposure to the CNS with a concomitant decrease in systemic drug levels generating a desirable distribution profile for CNS acting drugs.

Ester-to-amide rearrangement of ethanolamine-derived prodrugs of sobetirome with increased blood-brain barrier penetration.

Current therapeutic options for treating demyelinating disorders such as multiple sclerosis (MS) do not stimulate myelin repair, thus creating a clinical need for therapeutic agents that address axonal remyelination. Thyroid hormone is known to play an important role in promoting developmental myelination and repair, and CNS permeable thyromimetic agents could offer an increased therapeutic index compared to endogenous thyroid hormone. Sobetirome is a clinical stage thyromimetic that has been shown to have promising activity in preclinical models related to MS and X-linked adrenoleukodystrophy (X-ALD), a genetic disease that involves demyelination. Here we report a new series of sobetirome prodrugs containing ethanolamine-based promoieties that were found to undergo an intramolecular O,N acyl migration to form the pharmacologically relevant amide species. Several of these systemically administered prodrugs deliver more sobetirome to the brain compared to unmodified sobetirome. Pharmacokinetic properties of the parent drug sobetirome and amidoalcohol prodrug 3 are described and prodrug 3 was found to be more potent than sobetirome in target engagement in the brain from systemic dosing.

Sobetirome prodrug esters with enhanced blood-brain barrier permeability.

There is currently great interest in developing drugs that stimulate myelin repair for use in demyelinating diseases such as multiple sclerosis. Thyroid hormone plays a key role in stimulating myelination during development and also controls the expression of important genes involved in myelin repair in adults. Because endogenous thyroid hormone in excess lacks a generally useful therapeutic index, it is not used clinically for indications other than hormone replacement; however, selective thyromimetics such as sobetirome offer a therapeutic alternative. Sobetirome is the only clinical-stage thyromimetic that is known to cross the blood-brain-barrier (BBB) and we endeavored to increase the BBB permeability of sobetirome using a prodrug strategy. Ester prodrugs of sobetirome were prepared based on literature reports of improved BBB permeability with other carboxylic acid containing drugs and BBB permeability was assessed in vivo. One sobetirome prodrug, ethanolamine ester 11, was found to distribute more sobetirome to the brain compared to an equimolar peripheral dose of unmodified sobetirome. In addition to enhanced brain levels, prodrug 11 displayed lower sobetirome blood levels and a brain/serum ratio that was larger than that of unmodified sobetirome. Thus, these data indicate that an ester prodrug strategy applied to sobetirome can deliver increased concentrations of the active drug to the central nervous system (CNS), which may prove useful in the treatment of CNS disorders.

Efficacy of the Herpes Simplex Virus 2 (HSV-2) Glycoprotein D/AS04 Vaccine against Genital HSV-2 and HSV-1 Infection and Disease in the Cotton Rat Sigmodon hispidus Model.

UNLABELLED: Subunit vaccines based on the herpes simplex virus 2 (HSV-2) glycoprotein D (gD-2) have been the major focus of HSV-2 vaccine development for the past 2 decades. Based on the promising data generated in the guinea pig model, a formulation containing truncated gD-2, aluminum salt, and MPL (gD/AS04) advanced to clinical trials. The results of these trials, however, were unexpected, as the vaccine protected against HSV-1 infection but not against HSV-2. To address this discrepancy, we developed a Depot medroxyprogesterone acetate (DMPA)-treated cotton rat Sigmodon hispidus model of HSV-2 and HSV-1 genital infection. The severity of HSV-1 genital herpes was less than that of HSV-2 genital herpes in cotton rats, and yet the model allowed for comparative evaluation of gD/AS04 immunogenicity and efficacy. Cotton rats were intramuscularly vaccinated using a prime boost strategy with gD/AS04 (Simplirix vaccine) or control vaccine formulation (hepatitis B vaccine FENDrix) and subsequently challenged intravaginally with HSV-2 or HSV-1. The gD/AS04 vaccine was immunogenic in cotton rats and induced serum IgG directed against gD-2 and serum HSV-2 neutralizing antibodies but failed to efficiently protect against HSV-2 disease or to decrease the HSV-2 viral load. However, gD/AS04 significantly reduced vaginal titers of HSV-1 and better protected animals against HSV-1 compared to HSV-2 genital disease. The latter finding is generally consistent with the clinical outcome of the Herpevac trial of Simplirix. Passive transfer of serum from gD/AS04-immunized cotton rats conferred stronger protection against HSV-1 genital disease. These findings suggest the need for alternative vaccine strategies and the identification of new correlates of protection. IMPORTANCE: In spite of the high health burden of genital herpes, there is still no effective intervention against the disease. The significant gap in knowledge on genital herpes pathogenesis has been further highlighted by the recent failure of GSK HSV-2 vaccine Simplirix (gD/AS04) to protect humans against HSV-2 and the surprising finding that the vaccine protected against HSV-1 genital herpes instead. In this study, we report that gD/AS04 has higher efficacy against HSV-1 compared to HSV-2 genital herpes in the novel DMPA-synchronized cotton rat model of HSV-1 and HSV-2 infection. The findings help explain the results of the Simplirix trial.