Protein phosphatase 5 mediates lipid metabolism through reciprocal control of glucocorticoid receptor and peroxisome proliferator-activated receptor-γ (PPARγ).

Glucocorticoid receptor-α (GRα) and peroxisome proliferator-activated receptor-γ (PPARγ) regulate adipogenesis by controlling the balance between lipolysis and lipogenesis. Here, we show that protein phosphatase 5 (PP5), a nuclear receptor co-chaperone, reciprocally modulates the lipometabolic activities of GRα and PPARγ. Wild-type and PP5-deficient (KO) mouse embryonic fibroblast cells were used to show binding of PP5 to both GRα and PPARγ. In response to adipogenic stimuli, PP5-KO mouse embryonic fibroblast cells showed almost no lipid accumulation with reduced expression of adipogenic markers (aP2, CD36, and perilipin) and low fatty-acid synthase enzymatic activity. This was completely reversed following reintroduction of PP5. Loss of PP5 increased phosphorylation of GRα at serines 212 and 234 and elevated dexamethasone-induced activity at prolipolytic genes. In contrast, PPARγ in PP5-KO cells was hyperphosphorylated at serine 112 but had reduced rosiglitazone-induced activity at lipogenic genes. Expression of the S112A mutant rescued PPARγ transcriptional activity and lipid accumulation in PP5-KO cells pointing to Ser-112 as an important residue of PP5 action. This work identifies PP5 as a fulcrum point in nuclear receptor control of the lipolysis/lipogenesis equilibrium and as a potential target in the treatment of obesity.

Prenatal Imaging Findings Predict Obstructive Fetal Airways Requiring EXIT.

OBJECTIVE: Detection of fetal airway compromise through imaging raises the possible need for ex utero intrapartum treatment (EXIT) procedures. Despite EXIT procedures involving massive resource utilization and posing increased risk to the mother, decisions for EXIT are usually based on anecdotal experience. Our objectives were to analyze prenatal consultations with potential fetal airway obstruction for imaging and obstetric findings used to determine management strategy. METHODS: Retrospective chart review was performed for prenatal abnormal fetal airway consults between 2004-2019 at a quaternary pediatric facility. Data collected included demographics, imaging characteristics, delivery information, and airway management. Our primary outcome was EXIT performance and the secondary outcome was postnatal airway management. Fisher's exact test was used to compare management decisions, outcomes, and imaging findings. RESULTS: Thirty-seven patients met inclusion criteria. The most common diagnoses observed were lymphatic malformation, teratoma, and micrognathia. Of the imaging findings collected, only midline neck mass location was associated with EXIT procedure performance. Factors associated with invasive airway support at birth were mass-induced in-utero neck extension and neck vessel compression, polyhydramnios, and micrognathia. CONCLUSIONS: Multidisciplinary input and interpretation of prenatal imaging can guide management of fetal airway-related pathology. EXIT is an overall safe procedure and can decrease risk due to airway obstruction at birth. We identified in-utero neck extension, neck vessel compression, micrognathia, and polyhydramnios as better indicators of a need for invasive airways measures at birth and suggest use of these criteria in combination with clinical judgement when recommending EXIT. LEVEL OF EVIDENCE: 4 Laryngoscope, 131:E1357-E1362, 2021.

Neutron Therapy for High-Grade Salivary Carcinomas in the Adjuvant and Primary Treatment Setting.

OBJECTIVES/HYPOTHESIS: Our primary objective was to compare differences in survival of patients with high-grade salivary gland carcinomas (SGCs) receiving adjuvant neutron versus photon radiotherapy using a hospital-based national cohort and restricted mean survival time (RMST) analysis. Our secondary objective was to compare survival of similar patients treated with primary neutron versus photon radiation. STUDY DESIGN: Multicenter, retrospective population-based study of patients within the National Cancer Database from 2004 to 2014. METHODS: One thousand eight hundred forty-four patients were selected on diagnosis of high-grade parotid and submandibular malignancies. One thousand seven hundred seventy-seven patients receiving photon and 67 patients receiving neutron therapy were identified who met inclusion criteria. Patients were then categorized as having primary surgery with adjuvant radiation or primary radiation without prior surgery. Bivariate analysis was performed to assess for differences between groups, and RMST analysis was performed at 1-, 2-, and 5-year timepoints with controlling for available covariate data. RESULTS: There was no significant difference in RMST for patients receiving neutrons over photons at 1, 2, and 5 years in the adjuvant setting. Among patients undergoing primary radiotherapy, there was a difference in RMST of 2.29 months at 1 year and 5.05 months at 2 years for neutrons over photons, though this benefit was not observed at 5 years post-therapy. CONCLUSIONS: For patients with high grade SGCs undergoing adjuvant photon versus neutron radiotherapy, there was no difference in RMST. There was observed to be a significant difference in RMST at 1 and 2 years among patients undergoing primary neutron therapy of up to 5 months. Given the benefit observed with primary neutron therapy, it should be considered in both the primary and adjuvant treatment setting. LEVEL OF EVIDENCE: 4 Laryngoscope, 131:541-547, 2021.

Optimizing value in head and neck cancer free flap surgery.

PURPOSE OF REVIEW: Value has become an increasingly important topic in healthcare delivery as our systems attempt to deliver increased value to patients at lower costs. This review highlights research performed regarding value for head and neck cancer reconstruction in three evolving areas: care pathway development, virtual surgical planning (VSP), and free flap versus locoregional flap reconstruction. RECENT FINDINGS: Improvements in quality-driven patient care in head and neck free tissue transfer are possible in a number of areas. Care pathways and appropriate management of patients prone to comorbidities decrease hospital length of stay and readmission rates. Further, high-cost hospital stays partially driven by ICU admissions may be reduced by step-down units. Intraoperatively, VSP may reduce operative time in difficult cases and careful selection of free versus locoregional flap reconstruction may decrease cost, operative time, and complications after surgery. SUMMARY: Head and neck reconstruction is a costly endeavor both for the patient and the healthcare system. Careful consideration of practices which may improve outcomes for patients while maximizing efficiency is necessary in our changing healthcare landscape, and providers should identify areas for improvement in their own practices. Further study within the field of head and neck oncology that are specific and data-driven are necessary.

Dose-dependent enhancement of T-lymphocyte priming and CTL lysis following ionizing radiation in an engineered model of oral cancer.

OBJECTIVES: Determine if direct tumor cell cytotoxicity, antigen release, and susceptibility to T-lymphocyte killing following radiation treatment is dose-dependent. MATERIALS AND METHODS: Mouse oral cancer cells were engineered to express full-length ovalbumin as a model antigen. Tumor antigen release with uptake and cross presentation of antigen by antigen presenting cells with subsequent priming and expansion of antigen-specific T-lymphocytes following radiation was modeled in vitro and in vivo. T-lymphocyte mediated killing was measured following radiation treatment using a novel impedance-based cytotoxicity assay. RESULTS: Radiation treatment induced dose-dependent induction of executioner caspase activity and apoptosis in MOC1 cells. In vitro modeling of antigen release and T-lymphocyte priming demonstrated enhanced proliferation of OT-1 T-lymphocytes with 8Gy treatment of MOC1ova cells compared to 2Gy. This was validated in vivo following treatment of established MOC1ova tumors and adoptive transfer of antigen-specific T-lymphocytes. Using a novel impedance-based cytotoxicity assay, 8Gy enhanced tumor cell susceptibility to T-lymphocyte killing to a greater degree than 2Gy. CONCLUSION: In the context of using clinically-relevant doses of radiation treatment as an adjuvant for immunotherapy, 8Gy is superior to 2Gy for induction of antigen-specific immune responses and enhancing tumor cell susceptibility to T-lymphocyte killing. These findings have significant implications for the design of trials combining radiation and immunotherapy.

Anti-PD-L1 Efficacy Can Be Enhanced by Inhibition of Myeloid-Derived Suppressor Cells with a Selective Inhibitor of PI3Kδ/γ.

Checkpoint inhibitors are relatively inefficacious in head and neck cancers, despite an abundance of genetic alterations and a T-cell-inflamed phenotype. One significant barrier to efficacy may be the recruitment of myeloid-derived suppressor cells (MDSC) into the tumor microenvironment. Here we demonstrate functional inhibition of MDSC with IPI-145, an inhibitor of PI3Kδ and PI3Kγ isoforms, which enhances responses to PD-L1 blockade. Combination therapy induced CD8+ T lymphocyte-dependent primary tumor growth delay and prolonged survival only in T-cell-inflamed tumor models of head and neck cancers. However, higher doses of IPI-145 reversed the observed enhancement of anti-PD-L1 efficacy due to off-target suppression of the activity of tumor-infiltrating T lymphocytes. Together, our results offer a preclinical proof of concept for the low-dose use of isoform-specific PI3Kδ/γ inhibitors to suppress MDSC to enhance responses to immune checkpoint blockade. Cancer Res; 77(10); 2607-19. ©2017 AACR.

Pools of programmed death-ligand within the oral cavity tumor microenvironment: Variable alteration by targeted therapies.

BACKGROUND: Enhanced understanding of programmed death-ligand (PD-L) expression in oral cancer is important for establishing rational combinations of emerging immune checkpoint and molecular targeted therapies. METHODS: We assessed PD-L and interferon (IFN) expression in immunogenic murine oral cancer-1 (MOC1) and poorly immunogenic MOC2 cell models after treatment with mammalian target of rapamycin (mTOR) and MEK1/2 small molecule inhibitors in vitro and in vivo. RESULTS: PD-L1 but not PD-L2 is expressed on MOC1 and 2 cells and is type I and II IFN-dependent. PD-L1 is differentially expressed on cancer and endothelial cells and infiltrating myeloid-derived suppressor cells, macrophages, and regulatory T cells (Tregs) in highly and poorly immunogenic tumors. PD-L1 expression is variably altered after treatment with inhibitors in vivo, with an imperfect relationship to alterations in IFN levels in the tumor microenvironment. CONCLUSION: PD-L1 expressed on cancer and infiltrating immune cells is variably altered by targeted therapies and may, in part, reflect changes in tumor IFN. © 2016 Wiley Periodicals, Inc. Head Neck 38:1176-1186, 2016.

Established T Cell-Inflamed Tumors Rejected after Adaptive Resistance Was Reversed by Combination STING Activation and PD-1 Pathway Blockade.

Patients with head and neck squamous cell carcinoma harbor T cell-inflamed and non-T cell-inflamed tumors. Despite this, only 20% of patients respond to checkpoint inhibitor immunotherapy. Lack of induction of innate immunity through pattern-recognition receptors, such as the stimulator of interferon (IFN) genes (STING) receptor, may represent a significant barrier to the development of effective antitumor immunity. Here, we demonstrate robust control of a T cell-inflamed (MOC1), but not non-T cell-inflamed (MOC2), model of head and neck cancer by activation of the STING pathway with the synthetic cyclic dinucleotide RP,RP dithio-c-di-GMP. Rejection or durable tumor control of MOC1 tumors was dependent upon a functional STING receptor and CD8 T lymphocytes. STING activation resulted in increased tumor microenvironment type 1 and type 2 IFN and greater expression of PD-1 pathway components in vivo Established MOC1 tumors were rejected and distant tumors abscopally controlled, after adaptive immune resistance had been reversed by the addition of PD-L1 mAb. These findings suggest that PD-1 pathway blockade may reverse adaptive immune resistance following cyclic dinucleotide treatment, enhancing both local and systemic antitumor immunity. Cancer Immunol Res; 4(12); 1061-71. ©2016 AACR.

Enhanced Tumor Control with Combination mTOR and PD-L1 Inhibition in Syngeneic Oral Cavity Cancers.

Significant subsets of patients with oral cancer fail to respond to single-agent programmed death (PD) blockade. Syngeneic models of oral cancer were used to determine if blocking oncogenic signaling improved in vivo responses to PD-L1 monoclonal antibody (mAb). Anti-PD-L1 enhanced durable primary tumor control and survival when combined with mTOR (rapamycin), but not in combination with MEK inhibition (PD901) in immunogenic MOC1 tumors. Conversely, PD-L1 mAb did not enhance tumor control in poorly immunogenic MOC2 tumors. Rapamycin enhanced expansion of peripheral antigen-specific CD8 T cells and IFNγ production following ex vivo antigen stimulation. More CD8 T cells infiltrated and were activated after PD-L1 mAb treatment in mice with immunogenic MOC1 tumors, which were stable or increased by the addition of rapamycin, but suppressed when PD901 was added. Rapamycin increased IFNγ production capacity in peripheral and tumor-infiltrating CD8 T cells. In vivo antibody depletion revealed a CD8 T-cell-dependent, and not NK cell-dependent mechanism of tumor growth inhibition after treatment with rapamycin and PD-L1 mAb, ruling out significant effects from NK cell-mediated antibody-dependent cellular cytotoxicity. Rapamycin also enhanced IFNγ or PD-L1 mAb treatment-associated induction of MHC class I expression on MOC1 tumor cells, an effect abrogated by depleting infiltrating CD8 T cells from the tumor microenvironment. These data conflict with traditional views of rapamycin as a universal immunosuppressant, and when combined with evidence of enhanced antitumor activity with the combination of rapamycin and PD-L1 mAb, suggest that this treatment combination deserves careful evaluation in the clinical setting. Cancer Immunol Res; 4(7); 611-20. ©2016 AACR.

Chronic cough in children.

IMPORTANCE: Chronic cough is a common complaint among pediatric patients, but little information exists on the types of diagnoses in these patients and therapeutic outcomes. OBJECTIVE: To characterize pediatric patients with chronic cough presenting to otolaryngology clinics, identify common causes and treatments, and evaluate therapeutic outcomes. DESIGN, SETTING, AND PARTICIPANTS: In this retrospective analysis, all medical records for pediatric patients seen at 2 otolaryngology clinics at a single tertiary care academic medical center from January 2009 through June 2013 were searched for relevant diagnostic codes. Patients younger than 18 years presenting with chronic cough (cough of >4 weeks' duration) were selected for study; 58 patients met the inclusion criteria and were selected for analysis. INTERVENTIONS: Each patient was evaluated by the clinician via history, physical examination, and various tests and procedures, depending on the individual case, including flexible laryngoscopy, imaging tests, and/or airway evaluations. Each patient underwent treatment based on the working diagnosis. MAIN OUTCOMES AND MEASURES: Primary outcomes included final diagnosis, response to treatment (as indicated by resolution of cough after initial therapy), and diagnostic workup undergone. RESULTS: Among the 58 included patients, the 3 most common diagnoses were related to infection (n = 23; 34%), airway hyperreactivity (n = 14; 24%), or gastroesophageal reflux disease (n = 14; 24%). Initial response to treatment was observed in 83% of patients (n = 48), while the remaining 17% (n = 10) required further evaluation. All 10 patients for whom initial treatment failed (100%) had a diagnosis involving airway hyperreactivity (P < .001). In addition, chest radiography was more likely to be ordered (odds ratio [OR], 16.4; 95% CI, 1.91-140.8; P = .002) and to contain pertinent positive findings (OR, 12.8; 95% CI, 1.15-142.6; P = .04) in patients for whom treatment failed. CONCLUSIONS AND RELEVANCE: Chronic cough in the pediatric otolaryngology setting differs from the typical presentation in the primary care setting. The top 3 causes encountered by the practicing otolaryngologist in a pediatric patient are infection, airway hyperreactivity, and gastroesophageal reflux disease. If initial otolaryngologic treatment fails, chest radiography and early pulmonary consult are recommended.

mTOR and MEK1/2 inhibition differentially modulate tumor growth and the immune microenvironment in syngeneic models of oral cavity cancer.

We investigated the effects of mTOR and MEK1/2 inhibition on tumor growth and the tumor microenvironment in immunogenic and poorly immunogenic models of murine oral cancer. In vitro, rapamycin and PD901 inhibited signaling through expected downstream targets, but only PD901 reduced viability and altered function of MOC cells. Following transplantation of MOC cells into immune-competent mice, effects on both cancer and infiltrating immune cells were characterized following rapamycin and/or PD901 treatment for 21 days. In vivo, both rapamycin and PD901 inhibition reduced primary growth of established MOC tumors on treatment. Following withdrawal of PD901, rapid rebound of tumor growth limited survival, whereas durable tumor control was observed following rapamycin treatment in immunogenic MOC1 tumors despite more robust inhibition of oncogenic signaling by PD901. Characterization of the immune microenvironment revealed diminished infiltration and activation of antigen-specific CD8+ T-cells and other immune cells following PD901 but not rapamycin in immunogenic tumors. Subsequent in vitro T-cell assays validated robust inhibition of T-cell expansion and activation following MEK inhibition compared to mTOR inhibition. CD8 cell depletion abrogated rapamycin-induced primary tumor growth inhibition in MOC1 mice. These data have critical implications in the design of combination targeted and immune therapies in oral cancer.

Discovery of glucocorticoid receptor-beta in mice with a role in metabolism.

Glucocorticoid hormones control diverse physiological processes, including metabolism and immunity, by activating the major glucocorticoid receptor (GR) isoform, GRalpha. However, humans express an alternative isoform, human (h)GRbeta, that acts as an inhibitor of hGRalpha to produce a state of glucocorticoid resistance. Indeed, evidence exists that hGRbeta contributes to many diseases and resistance to glucocorticoid hormone therapy. However, rigorous testing of the GRbeta contribution has not been possible, because rodents, especially mice, are not thought to express the beta-isoform. Here, we report expression of GRbeta mRNA and protein in the mouse. The mGRbeta isoform arises from a distinct alternative splicing mechanism utilizing intron 8, rather than exon 9 as in humans. The splicing event produces a form of beta that is similar in structure and functionality to hGRbeta. Mouse (m)GRbeta has a degenerate C-terminal region that is the same size as hGRbeta. Using a variety of newly developed tools, such as a mGRbeta-specific antibody and constructs for overexpression and short hairpin RNA knockdown, we demonstrate that mGRbeta cannot bind dexamethasone agonist, is inhibitory of mGRalpha, and is up-regulated by inflammatory signals. These properties are the same as reported for hGRbeta. Additionally, novel data is presented that mGRbeta is involved in metabolism. When murine tissue culture cells are treated with insulin, no effect on mGRalpha expression was observed, but GRbeta was elevated. In mice subjected to fasting-refeeding, a large increase of GRbeta was seen in the liver, whereas mGRalpha was unchanged. This work uncovers the much-needed rodent model of GRbeta for investigations of physiology and disease.

Susceptibility to diet-induced hepatic steatosis and glucocorticoid resistance in FK506-binding protein 52-deficient mice.

Although FK506-binding protein 52 (FKBP52) is an established positive regulator of glucocorticoid receptor (GR) activity, an in vivo role for FKBP52 in glucocorticoid control of metabolism has not been reported. To address this question, FKBP52(+/-) mice were placed on a high-fat (HF) diet known to induce obesity, hepatic steatosis, and insulin resistance. Tissue profiling of wild-type mice showed high levels of FKBP52 in the liver but little to no expression in muscle or adipose tissue, predicting a restricted pattern of FKBP52 effects on metabolism. In response to HF, FKBP52(+/-) mice demonstrated a susceptibility to hyperglycemia and hyperinsulinemia that correlated with reduced insulin clearance and reduced expression of hepatic CEACAM1 (carcinoembryonic antigen-related cell adhesion molecule 1), a mediator of clearance. Livers of HF-fed mutant mice had high lipid content and elevated expression of lipogenic genes (peroxisome proliferator-activated receptor gamma, fatty acid synthase, and sterol regulatory element-binding protein 1c) and inflammatory markers (TNFalpha). Interestingly, mutant mice under HF showed elevated serum corticosterone, but their steatotic livers had reduced expression of gluconeogenic genes (phosphoenolpyruvate carboxy kinase, glucose 6 phosphatase, and pyruvate dehydrogenase kinase 4), whereas muscle and adipose expressed normal to elevated levels of glucocorticoid markers. These data suggest a state of glucocorticoid resistance arising from liver-specific loss of GR activity. Consistent with this hypothesis, reduced expression of gluconeogenic genes and CEACAM1 was observed in dexamethasone-treated FKBP52-deficient mouse embryonic fibroblast cells. We propose a model in which FKBP52 loss reduces GR control of gluconeogenesis, predisposing the liver to steatosis under HF-diet conditions attributable to a shunting of metabolism from glucose production to lipogenesis.