Tumor Cell Extrinsic Synaptogyrin 3 Expression as a Diagnostic and Prognostic Biomarker in Head and Neck Cancer.

Over 70% of oropharyngeal head and neck squamous cell carcinoma (HNSC) cases in the United States are positive for human papillomavirus (HPV) yet biomarkers for stratifying oropharyngeal head and neck squamous cell carcinoma (HNSC) patient risk are limited. We used immunogenomics to identify differentially expressed genes in immune cells of HPV(+) and HPV(-) squamous carcinomas. Candidate genes were tested in clinical specimens using both quantitative RT-PCR and IHC and validated by IHC using the Carolina Head and Neck Cancer Study (CHANCE) tissue microarray of HNSC cases. We performed multiplex immunofluorescent staining to confirm expression within the immune cells of HPV(+) tumors, receiver operating characteristic (ROC) curve analyses, and assessed survival outcomes. The neuronal gene Synaptogyrin-3 (SYNGR3) is robustly expressed in immune cells of HPV(+) squamous cancers. Multiplex immunostaining and single cell RNA-seq analyses confirmed SYNGR3 expression in T cells, but also unexpectedly in B cells of HPV(+) tumors. ROC curve analyses revealed that combining SYNGR3 and p16 provides more sensitivity and specificity for HPV detection compared to p16 IHC alone. SYNGR3-high HNSC patients have significantly better prognosis with five-year OS and DSS rates of 60% and 71%, respectively. Moreover, combining p16 localization and SYNGR3 expression can further risk stratify HPV(+) patients such that high cytoplasmic, low nuclear p16 do significantly worse (Hazard Ratio, 8.6; P = 0.032) compared to patients with high cytoplasmic, high nuclear p16. SYNGR3 expression in T and B cells is associated with HPV status and enhanced survival outcomes of HNSC patients.

Activation of the CREB Coactivator CRTC2 by Aberrant Mitogen Signaling promotes oncogenic functions in HPV16 positive head and neck cancer.

Head and neck squamous cell carcinoma (HNSCC) is the 6th most common cancer worldwide and incidence rates are continuing to rise globally. Patients often present with locally advanced disease and a staggering 50% chance of relapse following treatment. Aberrant activation of adaptive response signaling pathways, such as the cAMP/PKA pathway, induce an array of genes associated with known cancer pathways that promote tumorigenesis and drug resistance. We identified the cAMP Regulated Transcription Coactivator 2 (CRTC2) to be overexpressed and constitutively activated in HNSCCs and this confers poor prognosis. CRTCs are regulated through their subcellular localization and we show that CRTC2 is exclusively nuclear in HPV(+) HNSCC, thus constitutively active, due to non-canonical Mitogen-Activated Kinase Kinase 1 (MEKK1)-mediated activation via a MEKK1-p38 signaling axis. Loss-of-function and pharmacologic inhibition experiments decreased CRTC2/CREB transcriptional activity by reducing nuclear CRTC2 via nuclear import inhibition and/or by eviction of CRTC2 from the nucleus. This shift in localization was associated with decreased proliferation, migration, and invasion. Our results suggest that small molecules that inhibit nuclear CRTC2 and p38 activity may provide therapeutic benefit to patients with HPV(+) HNSCC.

TP53, CDKN2A/P16, and NFE2L2/NRF2 regulate the incidence of pure- and combined-small cell lung cancer in mice.

Studies have shown that Nrf2E79Q/+ is one of the most common mutations found in human tumors. To elucidate how this genetic change contributes to lung cancer, we compared lung tumor development in a genetically-engineered mouse model (GEMM) with dual Trp53/p16 loss, the most common mutations found in human lung tumors, in the presence or absence of Nrf2E79Q/+. Trp53/p16-deficient mice developed combined-small cell lung cancer (C-SCLC), a mixture of pure-SCLC (P-SCLC) and large cell neuroendocrine carcinoma. Mice possessing the LSL-Nrf2E79Q mutation showed no difference in the incidence or latency of C-SCLC compared with Nrf2+/+ mice. However, these tumors did not express NRF2 despite Cre-induced recombination of the LSL-Nrf2E79Q allele. Trp53/p16-deficient mice also developed P-SCLC, where activation of the NRF2E79Q mutation associated with a higher incidence of this tumor type. All C-SCLCs and P-SCLCs were positive for NE-markers, NKX1-2 (a lung cancer marker) and negative for P63 (a squamous cell marker), while only P-SCLC expressed NRF2 by immunohistochemistry. Analysis of a consensus NRF2 pathway signature in human NE+-lung tumors showed variable activation of NRF2 signaling. Our study characterizes the first GEMM that develops C-SCLC, a poorly-studied human cancer and implicates a role for NRF2 activation in SCLC development.

Lysyl hydroxylase 2-induced collagen cross-link switching promotes metastasis in head and neck squamous cell carcinomas.

Head and neck squamous cell carcinoma (HNSCC) is the 6th most common cancer worldwide and incidence rates are continuing to rise globally. HNSCC patient prognosis is closely related to the occurrence of tumor metastases, and collagen within the tumor microenvironment (TME) plays a key role in this process. Lysyl hydroxylase 2 (LH2), encoded by the Procollagen-Lysine,2-Oxoglutarate 5-Dioxygenase 2 (PLOD2) gene, catalyzes hydroxylation of telopeptidyl lysine (Lys) residues of fibrillar collagens which then undergo subsequent modifications to form stable intermolecular cross-links that change the biomechanical properties (i.e. quality) of the TME. While LH2-catalyzed collagen modification has been implicated in driving tumor progression and metastasis in diverse cancers, little is known about its role in HNSCC progression. Thus, using gain- and loss-of-function studies, we examined the effects of LH2 expression levels on collagen cross-linking and cell behavior in vitro and in vivo using a tractable bioluminescent imaging-based orthotopic xenograft model. We found that LH2 overexpression dramatically increases HNSCC cell migratory and invasive abilities in vitro and that LH2-driven changes in collagen cross-linking robustly induces metastasis in vivo. Specifically, the amount of LH2-mediated collagen cross-links increased significantly with PLOD2 overexpression, without affecting the total quantity of collagen cross-links. Conversely, LH2 knockdown significantly blunted HNSCC cells invasive capacity in vitro and metastatic potential in vivo. Thus, regardless of the total "quantity" of collagen crosslinks, it is the "quality" of these cross-links that is the key driver of HNSCC tumor metastatic dissemination. These data implicate LH2 as a key regulator of HNSCC tumor invasion and metastasis by modulating collagen cross-link quality and suggest that therapeutic strategies targeting LH2-mediated collagen cross-linking in the TME may be effective in controlling tumor progression and improving disease outcomes.

A conditional mouse expressing an activating mutation in NRF2 displays hyperplasia of the upper gastrointestinal tract and decreased white adipose tissue.

Activation of the nuclear factor (erythroid-derived 2)-like 2 (NFE2L2 or NRF2) transcription factor is a critical and evolutionarily conserved cellular response to oxidative stress, metabolic stress, and xenobiotic insult. Deficiency of NRF2 results in hypersensitivity to a variety of stressors, whereas its aberrant activation contributes to several cancer types, most commonly squamous cell carcinomas of the esophagus, oral cavity, bladder, and lung. Between 10% and 35% of patients with squamous cell carcinomas display hyperactive NRF2 signaling, harboring activating mutations and copy number amplifications of the NFE2L2 oncogene or inactivating mutations or deletions of KEAP1 or CUL3, the proteins of which co-complex to ubiquitylate and degrade NRF2 protein. To better understand the role of NRF2 in tumorigenesis and more broadly in development, we engineered the endogenous Nfe2l2 genomic locus to create a conditional mutant LSL-Nrf2E79Q mouse model. The E79Q mutation, one of the most commonly observed NRF2-activating mutations in human squamous cancers, codes for a mutant protein that does not undergo KEAP1/CUL3-dependent degradation, resulting in its constitutive activity. Expression of NRF2 E79Q protein in keratin 14 (KRT14)-positive murine tissues resulted in hyperplasia of squamous cell tissues of the tongue, forestomach, and esophagus, a stunted body axis, decreased weight, and decreased visceral adipose depots. RNA-seq profiling and follow-up validation studies of cultured NRF2E79Q murine esophageal epithelial cells revealed known and novel NRF2-regulated transcriptional programs, including genes associated with squamous cell carcinoma (e.g. Myc), lipid and cellular metabolism (Hk2, Ppard), and growth factors (Areg, Bmp6, Vegfa). These data suggest that in addition to decreasing adipogenesis, KRT14-restricted NRF2 activation drives hyperplasia of the esophagus, forestomach, and tongue, but not formation of squamous cell carcinoma. © 2020 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Gain-of-function genetic screen of the kinome reveals BRSK2 as an inhibitor of the NRF2 transcription factor.

Nuclear factor erythroid 2-related factor 2 (NFE2L2, also known as NRF2) is a transcription factor and master regulator of cellular antioxidant response. Aberrantly high NRF2-dependent transcription is recurrent in human cancer, but conversely NRF2 activity diminishes with age and in neurodegenerative and metabolic disorders. Although NRF2-activating drugs are clinically beneficial, NRF2 inhibitors do not yet exist. Here, we describe use of a gain-of-function genetic screen of the kinome to identify new druggable regulators of NRF2 signaling. We found that the under-studied protein kinase brain-specific kinase 2 (BRSK2) and the related BRSK1 kinases suppress NRF2-dependent transcription and NRF2 protein levels in an activity-dependent manner. Integrated phosphoproteomics and RNAseq studies revealed that BRSK2 drives 5'-AMP-activated protein kinase α2 (AMPK) signaling and suppresses the mTOR pathway. As a result, BRSK2 kinase activation suppresses ribosome-RNA complexes, global protein synthesis and NRF2 protein levels. Collectively, our data illuminate the BRSK2 and BRSK1 kinases, in part by functionally connecting them to NRF2 signaling and mTOR. This signaling axis might prove useful for therapeutically targeting NRF2 in human disease.This article has an associated First Person interview with the first author of the paper.

A piggyBac-based toolkit for inducible genome editing in mammalian cells.

We describe the development and application of a novel series of vectors that facilitate CRISPR-Cas9-mediated genome editing in mammalian cells, which we call CRISPR-Bac. CRISPR-Bac leverages the piggyBac transposon to randomly insert CRISPR-Cas9 components into mammalian genomes. In CRISPR-Bac, a single piggyBac cargo vector containing a doxycycline-inducible Cas9 or catalytically dead Cas9 (dCas9) variant and a gene conferring resistance to Hygromycin B is cotransfected with a plasmid expressing the piggyBac transposase. A second cargo vector, expressing a single-guide RNA (sgRNA) of interest, the reverse-tetracycline TransActivator (rtTA), and a gene conferring resistance to G418, is also cotransfected. Subsequent selection on Hygromycin B and G418 generates polyclonal cell populations that stably express Cas9, rtTA, and the sgRNA(s) of interest. We show that CRISPR-Bac can be used to knock down proteins of interest, to create targeted genetic deletions with high efficiency, and to activate or repress transcription of protein-coding genes and an imprinted long noncoding RNA. The ratio of sgRNA-to-Cas9-to-transposase can be adjusted in transfections to alter the average number of cargo insertions into the genome. sgRNAs targeting multiple genes can be inserted in a single transfection. CRISPR-Bac is a versatile platform for genome editing that simplifies the generation of mammalian cells that stably express the CRISPR-Cas9 machinery.

Mechanisms of exercise intolerance in heart failure with preserved ejection fraction: the role of abnormal peripheral oxygen extraction.

BACKGROUND: Exercise capacity as measured by peak oxygen uptake (Vo2) is similarly impaired in patients with heart failure with preserved ejection fraction (HFpEF) and heart failure with reduced ejection fraction (HFrEF). However, characterization of how each component of Vo2 changes in response to incremental exercise in HFpEF versus HFrEF has not been previously defined. We hypothesized that abnormally low peripheral o2 extraction (arterio-mixed venous o2 content difference, [C(a-v)o2]) during exercise significantly contributes to impaired exercise capacity in HFpEF. METHODS AND RESULTS: We performed maximum incremental cardiopulmonary exercise testing with invasive hemodynamic monitoring on 104 patients with symptomatic NYHA II to IV heart failure (HFpEF, n=48, peak Vo2=13.9±0.5 mL kg(-1) min(-1), mean±SEM, and HFrEF, n=56, peak Vo2=12.1±0.5 mL kg(-1) min(-1)) and 24 control subjects (peak Vo2 27.0±1.7 mL kg(-1) min(-1)). Peak exercise C(a-v)o2 was lower in HFpEF compared with HFrEF (11.5±0.27 versus 13.5±0.34 mL/dL, respectively, P<0.0001), despite no differences in age, hemoglobin level, peak respiratory exchange ratio, Cao2, or cardiac filling pressures. Peak C(a-v)o2 and peak heart rate emerged as the leading predictors of peak Vo2 in HFpEF. Impaired peripheral o2 extraction was the predominant limiting factor to exercise capacity in 40% of patients with HFpEF and was closely related to elevated systemic blood pressure during exercise (r=0.49, P=0.0005). CONCLUSIONS: In the first study to directly measure C(a-v)o2 throughout exercise in HFpEF, HFrEF, and normals, we found that peak C(a-v)o2 was a major determinant of exercise capacity in HFpEF. The important functional limitation imposed by impaired o2 extraction may reflect intrinsic abnormalities in skeletal muscle or peripheral microvascular function, and represents a potential target for therapeutic intervention.

Axial loading screw fixation for chevron type osteotomies of the distal first metatarsal: a retrospective outcomes analysis.

The distal chevron osteotomy is a widely accepted technique for the treatment of hallux abductovalgus deformity. Although the osteotomy is considered to be stable, displacements of the capital fragment has been described. We propose a new method for fixation of the osteotomy involving the axial loading screw (ALS) used in addition to single screw fixation. We believe this method will provide a more mechanically stable construct. We reviewed the charts of 46 patients in whom 52 feet underwent a distal chevron osteotomy that was fixated with either 1 screw or 2 screws that included the ALS. We hypothesized that the ALS group would have fewer displacements and would heal more quickly than the single screw fixation group. We found that the group with ALS fixation had healed at a mean of 6.5 weeks and that the group with single screw fixation had healed at 9.53 weeks (p = .001). Also, 8 cases occurred of displacement of the capital fragment in the single screw, control group compared with 2 cases of displacement in the ALS group. However, this finding was not statistically significant. The addition of the ALS to single screw fixation allowed the patients to heal approximately 3 weeks earlier than single screw fixation alone. The ALS is a fixation option for the surgeon to consider when osseous correction of hallux abducto valgus is performed.

Prolonged mean VO2 response time in systolic heart failure: an indicator of impaired right ventricular-pulmonary vascular function.

BACKGROUND: In patients with left ventricular systolic dysfunction (LVSD), the rate at which oxygen uptake (VO2) increases on initiation of exercise is inadequate to match metabolic demands. To gain mechanistic insights into delayed VO2 kinetics in LVSD, we simultaneously assessed hemodynamic measurements, ventilatory parameters, and peripheral oxygen usage during exercise. METHODS AND RESULTS: Forty-two patients with symptomatic LVSD (age, 59±2 years [mean±SEM]; LV ejection fraction, 30±1%) and 17 controls (LV ejection fraction, 68±1%) underwent maximum upright cycle ergometry cardiopulmonary exercise testing. Hemodynamic monitoring and first-pass radionuclide ventriculography were performed at rest and during exercise. VO2 kinetics were quantified by mean response time (MRT), which was significantly longer in patients with LVSD compared with controls (64±3 versus 45±5 s; P=0.004). In LVSD patients, MRT was associated with higher biventricular filling pressures and reduced cardiac output during early exercise. LVSD patients with MRT ≥60 s, compared with LVSD subjects with MRT <60 s, demonstrated greater impairment in right ventricular-pulmonary vascular function during exercise as evidenced by lower right ventricular ejection fraction (35±2 versus 45±2%; P=0.03), steeper increment in transpulmonary gradient relative to cardiac output (3.7 versus 2.2 mm Hg/L; P<0.001), and increased ventilatory dead-space fraction (17±1 versus 12±2%; P=0.03). In contrast, MRT was not associated with LV ejection fraction (rest, exercise), PaO2, hemoglobin, or resting pulmonary function test results. CONCLUSIONS: Delayed oxygen uptake on initiation of exercise (ie, MRT ≥60 s) in LVSD is closely related to impaired right ventricular-pulmonary vascular function and may represent an important surrogate for inability to augment RV performance during physical activity in patients with heart failure.

Exercise oscillatory ventilation in heart failure.

Irregular breathing characterized by cyclic variation of ventilation with a period of approximately 1 min has been recognized in patients with heart failure for almost two centuries. Periodic breathing during exercise is a noninvasive parameter that is easily recognizable during submaximal cardiopulmonary exercise testing. Recent studies have established that periodic breathing during exercise not only signals significant impairment in resting and exercise hemodynamic parameters but also potently predicts adverse events in heart failure patients. This article reviews the mechanistic basis of periodic breathing and the clinical utility of discerning patterns of irregular breathing in patients with heart failure.

Is colorectal surveillance indicated in patients with PTEN mutations?

AIM: Patients with germline phosphatase and tensin homologue (PTEN) mutations develop hamartomatous lesions in several organs and are at increased risk of various malignancies. We assessed the lifetime risk of benign and malignant gastrointestinal lesions in patients with a proven PTEN mutation. METHOD: Data on gender, mutation, dates of birth, last contact, and diagnosis, location and type of gastrointestinal lesions were collected from nine countries. The lifetime risk of gastrointestinal lesions was calculated by Kaplan-Meier methods. RESULTS: A total of 156 patients (67 men, 43%) from 101 families with a PTEN mutation were included. Patients were born between 1928 and 2008. Benign gastrointestinal polyps were reported in 49 (31%) patients at a mean age of 38 years (range 18-62 years) and were most often hamartomas. Twenty-two (44%) patients had upper as well as lower gastrointestinal lesions, 14 (29%) had only colonic lesions and 13 (27%) had gastrointestinal lesions at unknown sites. The cumulative risk of developing benign gastrointestinal polyps was 70% at age 60. Four patients (two men) developed colorectal carcinoma at 53, 57, 59 and 62 years, respectively. The cumulative risk of developing colorectal carcinoma was 18% at age 60. Except for one carcinoid in the small intestine, no upper gastrointestinal cancers were observed. CONCLUSION: Benign gastrointestinal lesions are common in PTEN mutation carriers, and a three- to four-fold increased lifetime risk of colorectal cancer compared with the general population may exist. Colorectal screening of patients with germline PTEN mutations is recommended, starting at age 40 years.

Exercise oscillatory ventilation in systolic heart failure: an indicator of impaired hemodynamic response to exercise.

BACKGROUND: Exercise oscillatory ventilation (EOV) is a noninvasive parameter that potently predicts outcomes in systolic heart failure (HF). However, mechanistic insights into EOV have been limited by the absence of studies relating EOV to invasive hemodynamic measurements and blood gases performed during exercise. METHODS AND RESULTS: Fifty-six patients with systolic HF (mean±SEM age, 59±2 years; left ventricular ejection fraction, 30±1%) and 19 age-matched control subjects were studied with incremental cardiopulmonary exercise testing. Fick cardiac outputs, filling pressures, and arterial blood gases were measured at 1-minute intervals during exercise. We detected EOV in 45% of HF (HF+EOV) patients and in none of the control subjects. The HF+EOV group did not differ from the HF patients without EOV (HF-EOV) in age, sex, body mass index, left ventricular ejection fraction, or origin of HF. Univariate predictors of the presence of EOV in HF, among measurements performed during exercise, included higher right atrial pressure and pulmonary capillary wedge pressure and lower cardiac index (CI) but not Paco2 or Pao2. Multivariate logistic regression identified that low exercise CI is the strongest predictor of EOV (odds ratio, 1.39 for each 1.0-L · min(-1) · m(-2) decrement in CI; 95% confidence interval, 1.14-1.70; P=0.001). Among HF patients with EOV, exercise CI was inversely related to EOV cycle length (R=-0.71) and amplitude (R=-0.60; both P<0.001). In 11 HF+EOV subjects treated with 12 weeks of sildenafil, EOV cycle length and amplitude decreased proportionately to increases in CI. CONCLUSION: Exercise oscillatory ventilation is closely related to reduced CI and elevated filling pressures during exercise and may be an important surrogate for exercise-induced hemodynamic impairment in HF patients. Clinical Trial Registration- URL: http://www.clinicaltrials.gov. Unique identifier: NCT00309790.

Pulmonary vascular response patterns during exercise in left ventricular systolic dysfunction predict exercise capacity and outcomes.

BACKGROUND: Elevated resting pulmonary arterial pressure (PAP) in patients with left ventricular systolic dysfunction (LVSD) purports a poor prognosis. However, PAP response patterns to exercise in LVSD and their relationship to functional capacity and outcomes have not been characterized. METHODS AND RESULTS: Sixty consecutive patients with LVSD (age 60±12 years, left ventricular ejection fraction 0.31±0.07, mean±SD) and 19 controls underwent maximum incremental cardiopulmonary exercise testing with simultaneous hemodynamic monitoring. During low-level exercise (30 W), LVSD subjects, compared with controls, had greater augmentation in mean PAPs (15±1 versus 5±1 mm Hg), transpulmonary gradients (5±1 versus 1±1 mm Hg), and effective pulmonary artery elastance (0.05±0.02 versus -0.03±0.01 mm Hg/mL, P<0.0001 for all). A linear increment in PAP relative to work (0.28±0.12 mm Hg/W) was observed in 65% of LVSD patients, which exceeded that observed in controls (0.07±0.02 mm Hg/W, P<0.0001). Exercise capacity and survival was worse in patients with a PAP/watt slope above the median than in patients with a lower slope. In the remaining 35% of LVSD patients, exercise induced a steep initial increment in PAP (0.41±0.16 mm Hg/W) followed by a plateau. The plateau pattern, compared with a linear pattern, was associated with reduced peak Vo(2) (10.6±2.6 versus 13.1±4.0 mL · kg(-1) · min(-1), P=0.005), lower right ventricular stroke work index augmentation with exercise (5.7±3.8 versus 9.7±5.0 g/m(2), P=0.002), and increased mortality (hazard ratio 8.1, 95% CI 2.7 to 23.8, P<0.001). CONCLUSIONS: A steep increment in PAP during exercise and failure to augment PAP throughout exercise are associated with decreased exercise capacity and survival in patients with LVSD, and may therefore represent therapeutic targets. CLINICAL TRIAL INFORMATION: URL: http://www.clinicaltrials.gov. Unique identifier: NCT00309790.