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While the role of mitochondrial metabolism in controlling T-lymphocyte activation and function is becoming more clear, the specifics of how mitochondrial redox signaling contributes to T-lymphocyte regulation remains elusive. Here, we examined the global effects of elevated mitochondrial superoxide (O2·-) on T-lymphocyte activation using a novel model of inducible manganese superoxide dismutase (MnSOD) knock-out. Loss of MnSOD led to specific increases in mitochondrial O2·- with no evident changes in hydrogen peroxide (H2O2), peroxynitrite (ONOO−), or copper/zinc superoxide dismutase (CuZnSOD) levels. Unexpectedly, both mitochondrial and glycolytic metabolism showed significant reductions in baseline, maximal capacities, and ATP production with increased mitochondrial O2·- levels. MnSOD knock-out T-lymphocytes demonstrated aberrant activation including widespread dysregulation in cytokine production and increased cellular apoptosis. Interestingly, an elevated proliferative signature defined by significant upregulation of cell cycle regulatory genes was also evident in MnSOD knock-out T-lymphocytes, but these cells did not show accelerated proliferative rates. Global disruption in T-lymphocyte DNA methylation and hydroxymethylation was also observed with increased mitochondrial O2·-, which was correlated to alterations in intracellular metabolite pools linked to the methionine cycle. Together, these results demonstrate a mitochondrial redox and metabolic couple that when disrupted may alter cellular processes necessary for proper T-lymphocyte activation. Keywords: Redox, Oxidative stress, Manganese superoxide dismutase, Immune, Adaptive immunity, Cytokines, Proliferation, Apoptosis, Metabolism, Methylation, Hydroxymethylation

Oral streptococci, key players in oral biofilm formation, are implicated in oral dysbiosis and various clinical conditions, including dental caries, gingivitis, periodontal disease, and oral cancer. Specifically, Streptococcus anginosus is associated with esophageal, gastric, and pharyngeal cancers, while Streptococcus mitis is linked to oral cancer. However, no study has investigated the mechanistic links between these Streptococcus species and cancer-related inflammatory responses. As an initial step, we probed the innate immune response triggered by S. anginosus and S. mitis in RAW264.7 macrophages. These bacteria exerted time- and dose-dependent effects on macrophage morphology without affecting cell viability. Compared with untreated macrophages, macrophages infected with S. anginosus exhibited a robust proinflammatory response characterized by significantly increased levels of inflammatory cytokines and mediators, including TNF, IL-6, IL-1β, NOS2, and COX2, accompanied by enhanced NF-κB activation. In contrast, S. mitis -infected macrophages failed to elicit a robust inflammatory response. Seahorse Xfe96 analysis revealed an increased extracellular acidification rate in macrophages infected with S. anginosus compared with S. mitis . At the 24-h time point, the presence of S. anginosus led to reduced extracellular itaconate, while S. mitis triggered increased itaconate levels, highlighting distinct metabolic profiles in macrophages during infection in contrast to aconitate decarboxylase expression observed at the 6-h time point. This initial investigation highlights how S. anginosus and S. mitis , two Gram-positive bacteria from the same genus, can prompt distinct immune responses and metabolic shifts in macrophages during infection.IMPORTANCEThe surge in head and neck cancer cases among individuals devoid of typical risk factors such as Human Papilloma Virus (HPV) infection and tobacco and alcohol use sparks an argumentative discussion around the emerging role of oral microbiota as a novel risk factor in oral squamous cell carcinoma (OSCC). While substantial research has dissected the gut microbiome's influence on physiology, the oral microbiome, notably oral streptococci, has been underappreciated during mucosal immunopathogenesis. Streptococcus anginosus , a viridans streptococci group, has been linked to abscess formation and an elevated presence in esophageal cancer and OSCC. The current study aims to probe the innate immune response to S. anginosus compared with the early colonizer Streptococcus mitis as an important first step toward understanding the impact of distinct oral Streptococcus species on the host immune response, which is an understudied determinant of OSCC development and progression., Competing Interests: The authors declare no conflict of interest.

Background: Anaplastic thyroid cancer (ATC) is uniformly lethal. BRAF V600E mutation is present in 45% of patients with ATC. Targeted therapy with combined BRAF and MEK inhibition in BRAF V600E -mutant ATC can be effective, but acquired resistance is common because this combination targets the same pathway. Drug matrix screening, in BRAF V600E ATC cells, of highly active compounds in combination with BRAF inhibition showed multitargeting tyrosine kinase inhibitors (MTKIs) had the highest synergistic/additive activity. Thus, we hypothesized that the combination of BRAF V600E inhibition and an MTKI is more effective than a single drug or combined BRAF and MEK inhibition in BRAF V600E -mutant ATC. We evaluated the effect of BRAF V600E inhibitors in combination with the MTKI axitinib and its mechanism(s) of action. Methods: We evaluated the effects of BRAF V600E inhibitors and axitinib alone and in combination in in vitro and in vivo models of BRAF V600E -mutant and wild-type ATC. Results: The combination of axitinib and BRAF V600E inhibitors (dabrafenib and PLX4720) showed an additive effect on inhibiting cell proliferation based on the Chou-Talalay algorithm in BRAF V600E -mutant ATC cell lines. This combination also significantly inhibited cell invasion and migration ( p  < 0.001) compared with the control. Dabrafenib and PLX4720 arrested ATC cells in the G0/G1 phase. Axitinib arrested ATC cells in the G2/M phase by decreasing phosphorylation of aurora kinase B (Thr232) and histone H3 (Ser10) proteins and by upregulating the c-JUN signaling pathway. The combination of BRAF inhibition and axitinib significantly inhibited tumor growth and was associated with improved survival in an orthotopic ATC model. Conclusions: The novel combination of axitinib and BRAF V600E inhibition enhanced anticancer activity in in vitro and in vivo models of BRAF V600E -mutant ATC. This combination may have clinical utility in BRAF V600E -mutant ATC that is refractory to current standard therapy, namely combined BRAF and MEK inhibition.

In this work, we propose a novel diagnostic biosensor that can enable stratification of disease states based on severity and hence allow for clear and actionable diagnoses. The scheme can potentially boost current Point-Of-Care (POC) biosensors for diseases that require time-critical stratification. Here, two key inflammatory biomarkers—Interleukin-8 and Interleukin-6—have been explored as proof of concept, and a four-class stratification of inflammatory disease severity is discussed. Our method is superior to traditional lab techniques as it is faster (<4 minutes turn-around time) and can work with any combination of disease biomarkers to categorize diseases by subtypes and severity. At its core, the biosensor relies on electrochemical impedance spectroscopy to transduce subtle inflammatory stimuli at the input for IL-8 and IL-6 for a limit of detection (LOD) of 1 pg/mL each. The biosensing scheme utilizes a two-stage random forest machine learning model for 4-state output disease classification with a 98.437% accuracy. This scheme can potentially boost the diagnostic power of current electrochemical biosensors for better precision therapy and improved patient outcomes.

Metabolic alterations regulate cancer aggressiveness and immune responses. Given the poor response of pancreatic ductal adenocarcinoma (PDAC) to conventional immunotherapies, we investigated the link between metabolic alterations and immunosuppression. Our metabolic enzyme screen indicated that elevated expression of CD73, an ecto-5'-nucleotidase that generates adenosine, correlates with increased aggressiveness. Correspondingly, we observed increased interstitial adenosine levels in tumors from spontaneous PDAC mouse models. Diminishing CD73 by genetic manipulations ablated in vivo tumor growth, and decreased myeloid-derived suppressor cells (MDSC) in orthotopic mouse models of PDAC. A high-throughput cytokine profiling demonstrated decreased GM-CSF in mice implanted with CD73 knockdowns. Furthermore, we noted increased IFN-γ expression by intratumoral CD4 + and CD8 + T cells in pancreatic tumors with CD73 knockdowns. Depletion of CD4 + T cells, but not CD8 + T cells abrogated the beneficial effects of decreased CD73. We also observed that splenic MDSCs from Nt5e knockdown tumor-bearing mice were incompetent in suppressing T cell activation in the ex vivo assays. Replenishing GM-CSF restored tumor growth in Nt5e knockout tumors, which was reverted by MDSC depletion. Finally, anti-CD73 antibody treatment significantly improved gemcitabine efficacy in orthotopic models. Thus, targeting the adenosine axis presents a novel therapeutic opportunity for improving the anti-tumoral immune response against PDAC., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

Pancreatic cancer (PC) is characterized by metabolic deregulations that often manifest as deviations in metabolite levels and aberrations in their corresponding metabolic genes across the clinical specimens and preclinical PC models. Cholesterol is one of the critical metabolites supporting PC, synthesized or acquired by PC cells. Nevertheless, the significance of the de novo cholesterol synthesis pathway has been controversial in PC, indicating the need to reassess this pathway in PC. We utilized preclinical models and clinical specimens of PC patients and cell lines and utilized mass spectrometry-based sterol analysis. Further, we also performed in silico analysis to corroborate the significance of de novo cholesterol synthesis pathway in PC. Our results demonstrated alteration in free sterol levels, including free cholesterol, across in vitro, in vivo, and clinical specimens of PC. Especially, our sterol analyses established consistent alterations in free cholesterol across the different PC models. Overall, this study demonstrates the significance and consistency in deviation of cholesterol synthesis pathway in PC while showing the aberrations in sterol metabolite intermediates and the related genes using preclinical models, in silico platforms, and the clinical specimens.

Background: Retrospective studies revealed that cigarette smoking enhances risk of incidence and worsens prognosis in pancreatic cancer (PC) patients. Poor prognosis in smoker cohort of PC patients indicates prevalence of cigarette smoke stimulated survival mechanisms yet to be explored in PC. In this study, cigarette smoke induced metabolic pathways were explored and targeted in PC., Methods: Human pancreatic ductal adenocarcinoma cell (PDAC) lines, genetically engineered mice models (GEMMs), mass spectrometry based heavy isotope-based metabolite analysis, cytotoxicity assays and Nuclear factor kappa-B (NF-kB) targeting were utilized in this study. Cigarette smoke extract (CSE) was prepared fresh each day by bubbling cell culture media with the smoke emitted from 85 mm, filtered, Code 1R6F reference cigarettes and used for in vitro procedures. High dose cigarette smoke exposure of GEMMs was achieved by daily exposure of animals to similar cigarettes, 6 h/day for a total period of 180 days., Findings: We observed that PDAC cells upregulate glutathione anabolism through cysteine uptake and glutamate cysteine ligase (GCLM), supporting survival, upon CSE exposure. In vivo, cigarette smoke exposure leads to concomitant upregulation of GCLM and activated NF-kB in the PDAC consistent with in vitro, in CSE-exposed PDAC. Finally, either inhibition of NF-kB or depletion of cysteine impaired PDAC cell survival in cigarette smoke exposed conditions through suppression of glutathione and ROS enhancement, reverted by glutathione supplementation., Interpretation: Our findings demonstrate scope for targeting smoke induced, NF-kB mediated, cysteine and glutathione metabolism for improving the survival of smoke addicted PDAC., (Copyright © 2021 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Background: Genetic testing for germline pheochromocytoma and paraganglioma susceptibility genes is associated with improved patient management. However, data are currently sparse on the probability of a positive testing result based on an individual's clinical presentation. This study evaluates clinical characteristics for association with testing positive for known pheochromocytoma and paraganglioma susceptibility genes., Methods: This retrospective analysis examined 111 patients with a diagnosis of pheochromocytoma and paraganglioma who underwent genetic testing. Logistic regression and receiver operating characteristic analyses were performed to identify factors associated with a positive genetic testing result. Probabilities were then calculated for combinations of significant factors to determine the likelihood of a positive test result in each group., Results: Of 32 patients with a family history of pheochromocytoma and paraganglioma, 31 (97%) had a germline mutation detected. Of 79 patients without a family history, 24 (30%) had a pathogenic germline mutation detected. In multivariate analysis, a positive family history, aged ≤47 years, and tumor size ≤2.9 cm were independent factors associated with a positive genetic testing result. Patients meeting all 3 criteria had a 100% probability compared with 13% in those without any of the criteria. In addition to a positive family history, having either aged ≤47 years or tumor size ≤2.9 cm resulted in a 90% and 100% probability of a positive result, respectively. In the absence of a family history, the probability in patients who were aged ≤47 years and had a tumor size ≤2.9 cm was 60%., Conclusion: In addition to a family history of pheochromocytoma and paraganglioma, aged ≤47 years, and tumor size ≤2.9 cm are associated with a higher probability of testing positive for a pheochromocytoma and paraganglioma susceptibility gene mutation. Patients meeting all 3 criteria have a 100% probability of a positive genetic testing result., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Background Anaplastic thyroid cancer (ATC) is aggressive with a poor prognosis, partly because of the immunosuppressive microenvironment created by tumor-associated macrophages (TAMs). Purpose To understand the relationship between TAM infiltration, tumor vascularization, and corresponding drug delivery by using ferumoxytol-enhanced MRI and macrin in an ATC mouse model. Materials and Methods ATC tumors were generated in 6-8-week-old female B6129SF1/J mice through intrathyroid injection to model orthotopic tumors, or intravenously to model hematogenous metastasis, and prospectively enrolled randomly into treatment cohorts ( n = 94 total; August 1, 2018, to January 15, 2020). Mice were treated with vehicle or combined serine/threonine-protein kinase B-Raf (BRAF) kinase inhibitor (BRAFi) and anti-PDL1 antibody (aPDL1). A subset was cotreated with therapies, including an approximately 70-nm model drug delivery nanoparticle (DDNP) to target TAM, and an antibody-neutralizing colony stimulating factor 1 receptor (CSF1R). Imaging was performed at the macroscopic level with ferumoxytol-MRI and microscopically with macrin. Genetically engineered Braf V600E/WT p53 -null allografts were used and complemented by a GFP-transgenic derivative and human xenografts. Tumor-bearing organs were processed by using tissue clearing and imaged with confocal microscopy and MRI. Two-tailed Wilcoxon tests were used for comparison (≥five per group). Results TAM levels were higher in orthotopic thyroid tumors compared with pulmonary metastatic lesions by 79% ± 23 (standard deviation; P < .001). These findings were concordant with ferumoxytol MRI, which showed 136% ± 88 higher uptake in thyroid lesions ( P = .02) compared with lung lesions. BRAFi and aPDL1 combination therapy resulted in higher tumor DDNP delivery by 39% ± 14 in pulmonary lesions ( P = .004). Compared with the untreated group, tumors following BRAFi, aPDL1, and CSF1R-blocking antibody combination therapy did not show greater levels of TAM or DDNP ( P = .82). Conclusion In a mouse model of anaplastic thyroid cancer, ferumoxytol MRI showed 136% ± 88 greater uptake in orthotopic thyroid tumors compared with pulmonary lesions, which reflected high vascularization and greater tumor-associated macrophage (TAM) levels. Serine/threonine-protein kinase B-Raf inhibitor and anti-programmed death ligand 1 antibody elicited higher local TAM levels and 43% ± 20 greater therapeutic nanoparticle delivery but not higher vascularization in pulmonary tumors. © RSNA, 2020 Online supplemental material is available for this article. See also the editorial by Luker in this issue.

Neuroblastoma are pediatric, extracranial malignancies showing alarming survival prognosis outcomes due to their resilience to current aggressive treatment regimens, including chemotherapies with cisplatin (CDDP) provided in the first line of therapy regimens. Metabolic deregulation supports tumor cell survival in drug-treated conditions. However, metabolic pathways underlying cisplatin-resistance are least studied in neuroblastoma. Our metabolomics analysis revealed that cisplatin-insensitive cells alter their metabolism; especially, the metabolism of amino acids was upregulated in cisplatin-insensitive cells compared to the cisplatin-sensitive neuroblastoma cell line. A significant increase in amino acid levels in cisplatin-insensitive cells led us to hypothesize that the mechanisms upregulating intracellular amino acid pools facilitate insensitivity in neuroblastoma. We hereby report that amino acid depletion reduces cell survival and cisplatin-insensitivity in neuroblastoma cells. Since cells regulate their amino acids levels through processes, such as autophagy, we evaluated the effects of hydroxychloroquine (HCQ), a terminal autophagy inhibitor, on the survival and amino acid metabolism of cisplatin-insensitive neuroblastoma cells. Our results demonstrate that combining HCQ with CDDP abrogated the amino acid metabolism in cisplatin-insensitive cells and sensitized neuroblastoma cells to sub-lethal doses of cisplatin. Our results suggest that targeting of amino acid replenishing mechanisms could be considered as a potential approach in developing combination therapies for treating neuroblastomas.

Background: The mechanistic target of rapamycin complex 1 (mTORC1) is a nutrient-sensing pathway and a key regulator of amino acid and glucose metabolism. Dysregulation of the mTOR pathways is implicated in the pathogenesis of metabolic syndrome, obesity, type 2 diabetes, and pancreatic cancer., Objectives: We investigated the impact of inhibition of mTORC1/mTORC2 and synergism with metformin on pancreatic tumor growth and metabolomics., Methods: Cell lines derived from pancreatic tumors of the KPC (Kras G12D/+ ; p53 R172H/+ ; Pdx1-Cre) transgenic mice model were implanted into the pancreas of C57BL/6 albino mice ( n  = 10/group). Two weeks later, the mice were injected intraperitoneally with daily doses of 1 ) Torin 2 (mTORC1/mTORC2 inhibitor) at a high concentration (TH), 2 ) Torin 2 at a low concentration (TL), 3 ) metformin at a low concentration (ML), 4 ) a combination of Torin 2 and metformin at low concentrations (TLML), or 5 ) DMSO vehicle (control) for 12 d. Tissues and blood samples were collected for targeted xenometabolomics analysis, drug concentration, and cell signaling., Results: Metabolomic analysis of the control and treated plasma samples showed differential metabolite profiles. Phenylalanine was significantly elevated in the TLML group compared with the control (+426%, P  = 0.0004), whereas uracil was significantly lower (-38%, P  = 0.009). The combination treatment reduced tumor growth in the orthotopic mouse model. TLML significantly decreased pancreatic tumor volume (498 ± 104 mm 3 ; 37%; P  < 0.0004) compared with control (1326 ± 134 mm 3 ; 100%), ML (853 ± 67 mm 3 ; 64%), TL (745 ± 167 mm 3 ; 54%), and TH (665 ± 182 mm 3 ; 50%) (ANOVA and post hoc tests). TLML significantly decreased tumor weights (0.66 ± 0.08 g; 52%) compared with the control (1.28 ± 0.19 g; 100%) ( P  < 0.002)., Conclusions: The combination of mTOR dual inhibition by Torin 2 and metformin is associated with an altered metabolomic profile and a significant reduction in pancreatic tumor burden compared with single-agent therapy, and it is better tolerated., (Copyright © The Author(s) on behalf of the American Society for Nutrition 2020.)

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

The regulation of the pleiotropic transcription factor, nuclear factor-κB (NF-κB) by miRNAs and proteins is extensively studied. More recently, the NF-κB signaling was also reported to be regulated by several long non-coding RNAs (lncRNAs) that constitute the major portion of the noncoding component of the human genome. The common NF-κB associated lncRNAs include NKILA, HOTAIR, MALAT1, ANRIL, Lethe, MIR31HG, and PACER. The lncRNA and NF-κB signaling crosstalk during cancer and other diseases such as cardiomyopathy, celiac disease, cerebral infarction, chronic kidney disease, diabetes mellitus, Kawasaki disease, pregnancy loss, and rheumatoid arthritis. Some NF-κB related lncRNAs can affect gene expression without modulating NF-κB signaling. Most of the lncRNAs with a potential to modulate NF-κB signaling are regulated by NF-κB itself suggesting a feedback regulation. The discovery of lncRNAs have provided a new type of regulation for the NF-κB signaling and thus could be explored for therapeutic interventions. The manner in which lncRNA and NF-κB crosstalk affects human pathophysiology is discussed in this review. The challenges associated with the therapeutic interventions of this crosstalk are also discussed., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Pancreatic cancer is the third leading cause of cancer-related deaths in the USA. Pancreatic tumors are characterized by enhanced glycolytic metabolism promoted by a hypoxic tumor microenvironment and a resultant acidic milieu. The metabolic reprogramming allows cancer cells to survive hostile microenvironments. Through the analysis of the principal metabolic pathways, we identified the specific metabolites that are altered during pancreatic cancer progression in the spontaneous progression (KPC) mouse model. Genetically engineered mice exhibited metabolic alterations during PanINs formation, even before the tumor development. To account for other cells in the tumor microenvironment and to focus on metabolic adaptations concerning tumorigenic cells only, we compared the metabolic profile of KPC and orthotopic tumors with those obtained from KPC-tumor derived cell lines. We observed significant upregulation of glycolysis and the pentose phosphate pathway metabolites even at the early stages of pathogenesis. Other biosynthetic pathways also demonstrated a few common perturbations. While some of the metabolic changes in tumor cells are not detectable in orthotopic and spontaneous tumors, a significant number of tumor cell-intrinsic metabolic alterations are readily detectable in the animal models. Overall, we identified that metabolic alterations in precancerous lesions are maintained during cancer development and are largely mirrored by cancer cells in culture conditions.

Acute myeloid leukemia (AML) is caused by abnormal production of white blood cells, red blood cells or platelets. The leukemia cells communicate with their microenvironment through nano-vesicle exosomes that are 30-100 nm in diameter. These nano-vesicles are released from body fluids upon fusion of an endocytic compartment with the cell membrane. Exosomes function as cargo to deliver signaling molecules to distant cells. This allows cross-talk between hematopoietic cells and other distant target cell environments. Exosomes support leukemia growth by acting as messengers between tumor cells and the microenvironment as well as inducing oncogenic factors such as c-Myc. Exosomes have also been used as biomarkers in the clinical diagnosis of leukemia. Glycogen synthase kinase-3 (GSK-3) and protein phosphatase 2A (PP2A) are two crucial signaling molecules involved in the AML pathogenesis and MYC stability. GSK-3 is a serine/threonine protein kinase that coordinates with over 40 different proteins during physiological/pathological conditions in blood cells. The dysregulation in GSK-3 has been reported during hematological malignancies. GSK-3 acts as a tumor suppressor by targeting c-MYC, MCL-1 and β-catenin. Conversely, GSK-3 can also act as tumor promoter in some instances. The pharmacological modulators of GSK-3 such as ABT-869, 6-Bromoindirubin-3'-oxime (BIO), GS-87 and LY2090314 have shown promise in the treatment of hematological malignancy. PP2A is a heterotrimeric serine/threonine phosphatase involved in the regulation of hematological malignancy. PP2A-activating drugs (PADs) can effectively antagonize leukemogenesis. The discovery of exosomes, kinase inhibitors and phosphatase activators have provided new hope to the leukemia patients. This review discusses the role of exosomes, GSK-3 and PP2A in the pathogenesis of leukemia. We provide evidence from both preclinical and clinical studies., (Copyright © 2019. Published by Elsevier Ltd.)

While the role of mitochondrial metabolism in controlling T-lymphocyte activation and function is becoming more clear, the specifics of how mitochondrial redox signaling contributes to T-lymphocyte regulation remains elusive. Here, we examined the global effects of elevated mitochondrial superoxide (O 2 - ) on T-lymphocyte activation using a novel model of inducible manganese superoxide dismutase (MnSOD) knock-out. Loss of MnSOD led to specific increases in mitochondrial O 2 - with no evident changes in hydrogen peroxide (H 2 O 2 ), peroxynitrite (ONOO - ), or copper/zinc superoxide dismutase (CuZnSOD) levels. Unexpectedly, both mitochondrial and glycolytic metabolism showed significant reductions in baseline, maximal capacities, and ATP production with increased mitochondrial O 2 - levels. MnSOD knock-out T-lymphocytes demonstrated aberrant activation including widespread dysregulation in cytokine production and increased cellular apoptosis. Interestingly, an elevated proliferative signature defined by significant upregulation of cell cycle regulatory genes was also evident in MnSOD knock-out T-lymphocytes, but these cells did not show accelerated proliferative rates. Global disruption in T-lymphocyte DNA methylation and hydroxymethylation was also observed with increased mitochondrial O 2 - , which was correlated to alterations in intracellular metabolite pools linked to the methionine cycle. Together, these results demonstrate a mitochondrial redox and metabolic couple that when disrupted may alter cellular processes necessary for proper T-lymphocyte activation., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

The deregulation of the actin cytoskeleton has been extensively studied in metastatic dissemination. However, the post-dissemination role of the actin cytoskeleton dysregulation is poorly understood. Here, we report that fascin, an actin-bundling protein, promotes lung cancer metastatic colonization by augmenting metabolic stress resistance and mitochondrial oxidative phosphorylation (OXPHOS). Fascin is directly recruited to mitochondria under metabolic stress to stabilize mitochondrial actin filaments (mtF-actin). Using unbiased metabolomics and proteomics approaches, we discovered that fascin-mediated mtF-actin remodeling promotes mitochondrial OXPHOS by increasing the biogenesis of respiratory Complex I. Mechanistically, fascin and mtF-actin control the homeostasis of mtDNA to promote mitochondrial OXPHOS. The disruption of mtF-actin abrogates fascin-mediated lung cancer metastasis. Conversely, restoration of mitochondrial respiration by using yeast NDI1 in fascin-depleted cancer cells is able to rescue lung metastasis. Our findings indicate that the dysregulated actin cytoskeleton in metastatic lung cancer could be targeted to rewire mitochondrial metabolism and to prevent metastatic recurrence., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

Patients with anaplastic thyroid cancer (ATC) have an extremely poor prognosis despite multimodal therapy with surgery and chemoradiation. Lenvatinib, a multi-targeted tyrosine kinase inhibitor, as well as checkpoint inhibitors targeting the programmed cell death pathway, have proven effective in some patients with advanced thyroid cancer. Combination of these therapies is a potential means to boost effectiveness and minimize treatment resistance in ATC. We utilized our novel immunocompetent murine model of orthotopic ATC to demonstrate that lenvatinib led to significant tumor shrinkage and increased survival, while combination therapy led to dramatic improvements in both. Lenvatinib monotherapy increased tumor-infiltrating macrophages, CD8 + T-cells, regulatory T-cells, and most notably, polymorphonuclear myeloid derived suppressor cells (PMN-MDSCs). While both combination therapies led to further increases in CD8 + T-cells, only the lenvatinib and anti-PD-1 combination decreased PMN-MDSCs. PMN-MDSC expansion was also seen in the blood of mice and one patient receiving lenvatinib therapy for ATC. RNA-Seq of the ATC cell line used in our mouse model demonstrated that lenvatinib has multifaceted effects on angiogenesis, response to hypoxia, the epithelial-to-mesenchymal transition, and on multiple pathways implicated in inflammation and host immunity. Combination of lenvatinib with anti-Gr-1 antibody ameliorated lenvatinib's expansion of MDSCs and significantly improved lenvatinib's anti-tumor effect. These data suggest that MDSCs play a negative role in ATC's response to lenvatinib and support future study of their role as a potential biomarker and treatment target., (© 2018 UICC.)

Pancreatic ductal adenocarcinoma (PDAC) is characterized by KRAS- and autophagy-dependent tumorigenic growth, but the role of KRAS in supporting autophagy has not been established. We show that, to our surprise, suppression of KRAS increased autophagic flux, as did pharmacological inhibition of its effector ERK MAPK. Furthermore, we demonstrate that either KRAS suppression or ERK inhibition decreased both glycolytic and mitochondrial functions. We speculated that ERK inhibition might thus enhance PDAC dependence on autophagy, in part by impairing other KRAS- or ERK-driven metabolic processes. Accordingly, we found that the autophagy inhibitor chloroquine and genetic or pharmacologic inhibition of specific autophagy regulators synergistically enhanced the ability of ERK inhibitors to mediate antitumor activity in KRAS-driven PDAC. We conclude that combinations of pharmacologic inhibitors that concurrently block both ERK MAPK and autophagic processes that are upregulated in response to ERK inhibition may be effective treatments for PDAC.

Isocitrate dehydrogenase 1 (IDH1) is the most commonly mutated metabolic enzyme in human malignancy. A heterozygous genetic alteration, arginine 132, promotes the conversion of α-ketoglutarate to D-2-hydroxyglutarate (2-HG). Although pharmacologic inhibitors of mutant IDH1 are promising, resistance mechanisms to targeted therapy are not understood. Additionally, the role of wild-type IDH1 (WT.IDH1) in cancer requires further study. Recently, it was observed that the regulatory RNA-binding protein, HuR (ELAVL1), protects nutrient-deprived cancer cells without IDH1 mutations, by stabilizing WT.IDH1 transcripts. In the present study, a similar regulatory effect on both mutant (Mut.IDH1) and WT.IDH1 transcripts in heterozygous IDH1-mutant tumors is observed. In ribonucleoprotein immunoprecipitation assays of IDH1-mutant cell lines, wild-type and mutant IDH1 mRNAs each bound to HuR. Both isoforms were profoundly downregulated at the mRNA and protein levels after genetic suppression of HuR (siRNAs or CRISPR deletion) in HT1080 (R132C IDH1 mutation) and BT054 cells (R132H). Proliferation and invasion were adversely affected after HuR suppression and metabolomic studies revealed a reduction in Pentose Phosphate Pathway metabolites, nucleotide precursors, and 2-HG levels. HuR-deficient cells were especially sensitive to stress, including low glucose conditions or a mutant IDH1 inhibitor (AGI-5198). IDH1-mutant cancer cells were rescued by WT.IDH1 overexpression to a greater extent than Mut.IDH1 overexpression under these conditions. This study reveals the importance of HuR's regulation of both mutant and wild-type IDH1 in tumors harboring a heterozygous IDH1 mutation with implications for therapy. IMPLICATIONS: This study highlights the HuR-IDH1 (mutant and wild-type IDH1) regulatory axis as a critical, actionable therapeutic target in IDH1-mutated cancer, and incomplete blockade of the entire HuR-IDH1 survival axis would likely diminish the efficacy of drugs that selectively target only the mutant isoenzyme., (©2018 American Association for Cancer Research.)

Background: The BRAF V600E mutation is the most common somatic mutation in thyroid cancer. The mechanism associated with BRAF-mutant tumor aggressiveness remains unclear. Lysyl oxidase (LOX) is highly expressed in aggressive thyroid cancers, and involved in cancer metastasis. The objective was to determine whether LOX mediates the effect of the activated MAPK pathway in thyroid cancer., Methods: The prognostic value of LOX and its association with mutated BRAF was analyzed in The Cancer Genome Atlas and an independent cohort. Inhibition of mutant BRAF and the MAPK pathway, and overexpression of mutant BRAF and mouse models of BRAF V600E were used to test the effect on LOX expression., Results: In The Cancer Genome Atlas cohort, LOX expression was higher in BRAF-mutant tumors compared to wild-type tumors (p < 0.0001). Patients with BRAF-mutant tumors with high LOX expression had a shorter disease-free survival (p = 0.03) compared to patients with a BRAF mutation and the low LOX group. In the independent cohort, a significant positive correlation between LOX and percentage of BRAF mutated cells was found. The independent cohort confirmed high LOX expression to be associated with a shorter disease-free survival (p = 0.01). Inhibition of BRAF V600E and MEK decreased LOX expression. Conversely, overexpression of mutant BRAF increased LOX expression. The mice with thyroid-specific expression of BRAF V600E showed strong LOX and p-ERK expression in tumor tissue. Inhibition of BRAF V600E in transgenic and orthotopic mouse models significantly reduced the tumor burden as well as LOX and p-ERK expression., Conclusions: The data suggest that BRAF V600E tumors with high LOX expression are associated with more aggressive disease. The biological underpinnings of the clinical findings were confirmed by showing that BRAF and the MAPK pathway regulate LOX expression.

Metabolic reprograming is an established hallmark of cancer cells. Pancreatic cancer cells, by virtue of the underlying oncogenic drivers, demonstrate metabolic reprograming to sustain growth, invasiveness, and therapy resistance. The increased demands of the growing tumor cells alter the metabolic and signaling pathways to meet the growing nutrient requirements. Investigating the metabolic vulnerabilities of tumor cells can help in developing effective therapeutics to target pancreatic cancer. In this chapter, we explain in detail the methods to evaluate the metabolic changes occurring in the tumor. This includes the glucose/glutamine uptake assays and the measurement of reactive oxygen species, extracellular acidification rate, and oxygen consumption rate in the tumor cells. All these physiological assays help in understanding the metabolic nature of the tumor.

Background: Patients with anaplastic thyroid cancer (ATC) have an extremely poor prognosis despite aggressive multimodal therapy. ATC has a high prevalence of BRAF V600E mutations and is associated with an immunosuppressive microenvironment; we previously demonstrated that the combination of BRAF inhibitor and checkpoint inhibitor immunotherapy synergistically reduce tumour volume in an immunocompetent mouse model of orthotopic ATC., Methods: We again utilised our mouse model of ATC to assess the combination of BRAF V600E inhibitor PLX4720 and anti-PD-L1 or anti-PD-1 antibody on survival, and performed immune cell profiling of lymphoid and myeloid-lineage cells during maximal treatment response and tumour regrowth., Results: Combination therapy dramatically improved mouse survival. Maximal tumour reduction was associated with increases in the number and cytotoxicity of CD8 + T cells and NK cells, as well as increases in mostly M1-polarised tumour-associated macrophages (TAM) and decreases in myeloid-derived suppressor-like cells. Regrowth of tumour occurred after 2-3 weeks of ongoing combination therapy, and was most significantly associated with decreased TAMs and a dramatic increase in M2-polarisation., Conclusions: Combination of PLX4720 and anti-PD-L1/PD-1 antibody dramatically reduced tumour volume, prolonged survival and improved the anti-tumour immune profile in murine ATC. Tumour growth inevitably recurred and demonstrated re-emergence of an immunosuppressive tumour microenvironment.

Background: BRAF V600E is the most common mutation in papillary thyroid carcinoma (PTC) and can be associated with aggressive disease. Previously, a highly sensitive blood RNA-based BRAF V600E assay was reported. The objective of this study was to assess the correlation of BRAF V600E circulating tumor RNA levels with surgical and medical treatment., Methods: Circulating BRAF V600E levels were assessed in (i) a murine model of undifferentiated (anaplastic) thyroid carcinoma with known BRAF V600E mutation undergoing BRAF V600E -inhibitor (BRAFi) treatment, and (ii) in 111 patients enrolled prior to thyroidectomy (n = 86) or treatment of advanced recurrent or metastatic PTC (n = 25). Blood samples were drawn for BRAF V600E analysis before and after treatment. Testing characteristics were assessed and positivity criteria optimized. Changes in blood BRAF V600E values were assessed and compared to clinical characteristics and response to therapy., Results: In a murine model of anaplastic thyroid carcinoma with BRAF V600E mutation, blood BRAF V600E RNA correlated with tumor volume in animals treated with BRAFi. In tissue BRAF V600E -positive (n = 36) patients undergoing initial surgery for PTC, blood BRAF V600E levels declined postoperatively (median 370.0-178.5 fg/ng; p = 0.002). In four patients with metastatic or poorly differentiated thyroid carcinoma receiving targeted therapies, blood BRAF V600E declined following therapy and corresponded with radiographic evidence of partial response or stable disease., Conclusions: This study shows the correlation of blood BRAF V600E levels in response to treatment in both an established animal model of thyroid cancer and in patients with BRAF V600E -positive tumors with all stages of disease. This assay represents an alternative biomarker in patients with positive thyroglobulin antibodies, and tumors, which do not express thyroglobulin.

Poorly differentiated thyroid carcinoma (PDTC) is an aggressive form of thyroid cancer that currently has limited effective treatment options. Immune checkpoint inhibitors (ICIs) have shown to be an effective treatment for a variety of carcinomas. In this study, we explore whether immune checkpoint pathways, such as programmed cell death ligand 1 (PD-L1) and indoleamine 2,3-dioxygenase 1 (IDO1), are activated in a cohort of patients with PDTC to determine whether ICIs may be an effective therapy for these patients. PDTC from 28 patients were stained for IDO1, PD-L1, and CD8 using immunohistochemistry. Staining was scored using an H-score, and PD-L1 and IDO1 expression was correlated with clinicopathologic characteristics. Positivity for PD-L1 and IDO1 was set at an H-score cutoff of five. Twenty-five percent (n = 7/28) of the PDTC were positive for PD-L1 expression. Twenty-nine percent (n = 2/7) of the PD-L1 positive PDTCs also co-expressed IDO1. The expression of PD-L1 in PDTC was significantly associated with tumor size and multifocality, with a non-significant trend towards associations with older age, extrathyroidal extension, presence of metastasis, higher stage, increased number of CD8+ T cells, and decreased disease-free and overall survival. PD-L1 expression occurs in a subset of PDTC, and is associated with a subset of clinical features of aggressive thyroid disease. Given the limited effective treatments for this patient population, consideration for ICIs as monotherapy or in combination with an IDO1 inhibitor should be explored as a novel treatment modality for patients with PDTC.

Ex vivo systems that incorporate features of the tumor microenvironment and model the dynamic response to immune checkpoint blockade (ICB) may facilitate efforts in precision immuno-oncology and the development of effective combination therapies. Here, we demonstrate the ability to interrogate ex vivo response to ICB using murine- and patient-derived organotypic tumor spheroids (MDOTS/PDOTS). MDOTS/PDOTS isolated from mouse and human tumors retain autologous lymphoid and myeloid cell populations and respond to ICB in short-term three-dimensional microfluidic culture. Response and resistance to ICB was recapitulated using MDOTS derived from established immunocompetent mouse tumor models. MDOTS profiling demonstrated that TBK1/IKKε inhibition enhanced response to PD-1 blockade, which effectively predicted tumor response in vivo Systematic profiling of secreted cytokines in PDOTS captured key features associated with response and resistance to PD-1 blockade. Thus, MDOTS/PDOTS profiling represents a novel platform to evaluate ICB using established murine models as well as clinically relevant patient specimens. Significance: Resistance to PD-1 blockade remains a challenge for many patients, and biomarkers to guide treatment are lacking. Here, we demonstrate feasibility of ex vivo profiling of PD-1 blockade to interrogate the tumor immune microenvironment, develop therapeutic combinations, and facilitate precision immuno-oncology efforts. Cancer Discov; 8(2); 196-215. ©2017 AACR. See related commentary by Balko and Sosman, p. 143 See related article by Deng et al., p. 216 This article is highlighted in the In This Issue feature, p. 127 ., (©2017 American Association for Cancer Research.)

Immune checkpoint blockade, exemplified by antibodies targeting the PD-1 receptor, can induce durable tumor regressions in some patients. To enhance the efficacy of existing immunotherapies, we screened for small molecules capable of increasing the activity of T cells suppressed by PD-1. Here, we show that short-term exposure to small-molecule inhibitors of cyclin-dependent kinases 4 and 6 (CDK4/6) significantly enhances T-cell activation, contributing to antitumor effects in vivo , due in part to the derepression of NFAT family proteins and their target genes, critical regulators of T-cell function. Although CDK4/6 inhibitors decrease T-cell proliferation, they increase tumor infiltration and activation of effector T cells. Moreover, CDK4/6 inhibition augments the response to PD-1 blockade in a novel ex vivo organotypic tumor spheroid culture system and in multiple in vivo murine syngeneic models, thereby providing a rationale for combining CDK4/6 inhibitors and immunotherapies. Significance: Our results define previously unrecognized immunomodulatory functions of CDK4/6 and suggest that combining CDK4/6 inhibitors with immune checkpoint blockade may increase treatment efficacy in patients. Furthermore, our study highlights the critical importance of identifying complementary strategies to improve the efficacy of immunotherapy for patients with cancer. Cancer Discov; 8(2); 216-33. ©2017 AACR. See related commentary by Balko and Sosman, p. 143 See related article by Jenkins et al., p. 196 This article is highlighted in the In This Issue feature, p. 127 ., (©2017 American Association for Cancer Research.)

Hypoxic conditions in the pancreatic tumor microenvironment lead to the stabilization of hypoxia-inducible factor-1 alpha (HIF-1α), which acts as the master regulator of cancer cell metabolism. HIF-1α-mediated metabolic reprogramming results in large-scale metabolite perturbations. Characterization of the metabolic intermediates and the corresponding metabolic pathways altered by HIF-1α would facilitate the identification of therapeutic targets for hypoxic microenvironments prevalent in pancreatic ductal adenocarcinoma and other solid tumors. Targeted metabolomic approaches are versatile in quantifying multiple metabolite levels in a single platform and, thus, enable the characterization of multiple metabolite alterations regulated by HIF-1α. In this chapter, we describe a detailed metabolomic approach for characterizing the hypoxia-induced metabolomic alterations using pancreatic cancer cell lines cultured in normoxic and hypoxic conditions. We elaborate the methodology of cell culture, hypoxic exposure, metabolite extraction, and relative quantification of polar metabolites from normoxia- and hypoxia-exposed cell extracts, using a liquid chromatography-coupled tandem mass spectrometry approach. Herein, using our metabolomic data, we also present the methods for metabolomic data representation.

Pancreatic adenocarcinoma is moderately responsive to gemcitabine-based chemotherapy, the most widely used single-agent therapy for pancreatic cancer. Although the prognosis in pancreatic cancer remains grim in part due to poor response to therapy, previous attempts at identifying and targeting the resistance mechanisms have not been very successful. By leveraging The Cancer Genome Atlas dataset, we identified lipid metabolism as the metabolic pathway that most significantly correlated with poor gemcitabine response in pancreatic cancer patients. Furthermore, we investigated the relationship between alterations in lipogenesis pathway and gemcitabine resistance by utilizing tissues from the genetically engineered mouse model and human pancreatic cancer patients. We observed a significant increase in fatty acid synthase (FASN) expression with increasing disease progression in spontaneous pancreatic cancer mouse model, and a correlation of high FASN expression with poor survival in patients and poor gemcitabine responsiveness in cell lines. We observed a synergistic effect of FASN inhibitors with gemcitabine in pancreatic cancer cells in culture and orthotopic implantation models. Combination of gemcitabine and the FASN inhibitor orlistat significantly diminished stemness, in part due to induction of endoplasmic reticulum (ER) stress that resulted in apoptosis. Moreover, direct induction of ER stress with thapsigargin caused a similar decrease in stemness and showed synergistic activity with gemcitabine. Our in vivo studies with orthotopic implantation models demonstrated a robust increase in gemcitabine responsiveness upon inhibition of fatty acid biosynthesis with orlistat. Altogether, we demonstrate that fatty acid biosynthesis pathway manipulation can help overcome the gemcitabine resistance in pancreatic cancer by regulating ER stress and stemness. Cancer Res; 77(20); 5503-17. ©2017 AACR ., (©2017 American Association for Cancer Research.)

Pancreatic cancer cells overexpressing Mucin 1 (MUC1) rely on aerobic glycolysis and, correspondingly, are dependent on glucose for survival. Our NMR metabolomics comparative analysis of control (S2-013.Neo) and MUC1-overexpressing (S2-013.MUC1) cells demonstrates that MUC1 reprograms glutamine metabolism upon glucose limitation. The observed alteration in glutamine metabolism under glucose limitation was accompanied by a relative decrease in the proliferation of MUC1-overexpressing cells compared with steady-state conditions. Moreover, glucose limitation induces G1 phase arrest where S2-013.MUC1 cells fail to enter S phase and synthesize DNA because of a significant disruption in pyrimidine nucleotide biosynthesis. Our metabolomics analysis indicates that glutamine is the major source of oxaloacetate in S2-013.Neo and S2-013.MUC1 cells, where oxaloacetate is converted to aspartate, an important metabolite for pyrimidine nucleotide biosynthesis. However, glucose limitation impedes the flow of glutamine carbons into the pyrimidine nucleotide rings and instead leads to a significant accumulation of glutamine-derived aspartate in S2-013.MUC1 cells.

Purpose: MUC1 , an oncogene overexpressed in multiple solid tumors, including pancreatic cancer, reduces overall survival and imparts resistance to radiation and chemotherapies. We previously identified that MUC1 facilitates growth-promoting metabolic alterations in pancreatic cancer cells. The present study investigates the role of MUC1-mediated metabolism in radiation resistance of pancreatic cancer by utilizing cell lines and in vivo models. Experimental Design: We used MUC1 -knockdown and -overexpressed cell line models for evaluating the role of MUC1-mediated metabolism in radiation resistance through in vitro cytotoxicity, clonogenicity, DNA damage response, and metabolomic evaluations. We also investigated whether inhibition of glycolysis could revert MUC1-mediated metabolic alterations and radiation resistance by using in vitro and in vivo models. Result s: MUC1 expression diminished radiation-induced cytotoxicity and DNA damage in pancreatic cancer cells by enhancing glycolysis, pentose phosphate pathway, and nucleotide biosynthesis. Such metabolic reprogramming resulted in high nucleotide pools and radiation resistance in in vitro models. Pretreatment with the glycolysis inhibitor 3-bromopyruvate abrogated MUC1-mediated radiation resistance both in vitro and in vivo , by reducing glucose flux into nucleotide biosynthetic pathways and enhancing DNA damage, which could again be reversed by pretreatment with nucleoside pools. Conclusions: MUC1-mediated nucleotide metabolism plays a key role in facilitating radiation resistance in pancreatic cancer and targeted effectively through glycolytic inhibition. Clin Cancer Res; 23(19); 5881-91. ©2017 AACR ., (©2017 American Association for Cancer Research.)

The increased rate of glycolysis and reduced oxidative metabolism are the principal biochemical phenotypes observed in pancreatic ductal adenocarcinoma (PDAC) that lead to the development of an acidic tumor microenvironment. The pH of most epithelial cell-derived tumors is reported to be lower than that of plasma. However, little is known regarding the physiology and metabolism of cancer cells enduring chronic acidosis. Here, we cultured PDAC cells in chronic acidosis (pH 6.9-7.0) and observed that cells cultured in low pH had reduced clonogenic capacity. However, our physiological and metabolomics analysis showed that cells in low pH deviate from glycolytic metabolism and rely more on oxidative metabolism. The increased expression of the transaminase enzyme GOT1 fuels oxidative metabolism of cells cultured in low pH by enhancing the non-canonical glutamine metabolic pathway. Survival in low pH is reduced upon depletion of GOT1 due to increased intracellular ROS levels. Thus, GOT1 plays an important role in energy metabolism and ROS balance in chronic acidosis stress. Our studies suggest that targeting anaplerotic glutamine metabolism may serve as an important therapeutic target in PDAC., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Bcl2 family proteins play an important role in the resistance of thyroid cancer cells to apoptosis induced by chemotherapeutic drugs and targeted therapies. BH3-profiling of seven fresh primary papillary thyroid cancer (PTC) tumors showed dependence for survival on Bcl-xL (2/7), Bcl2 (2/7), and Mcl-1 (2/7), while the majority of thyroid cell lines were mainly dependent on Bcl-xL. Targeting Bcl2 family proteins with the BH3 mimetic, ABT-737, while simultaneously inhibiting ERK pathway proteins with PLX4720 and PD325901 was shown to induce significantly high apoptosis in the majority of cell lines (8505c, SW1736, HTh7, BCPAP) and moderate apoptosis in the TPC-1 cell line. In orthotopic thyroid cancer mouse models of 8505c and BCPAP, treatment with the triple drug combination reduced the size of the tumors and showed significantly higher numbers of cells undergoing apoptosis. This treatment increased the expression of pro-apoptotic protein Bim, while decreasing anti-apoptotic protein Mcl-1. Our results suggest that analyzing the results of BH3-profiling along with the mutational status of tumor can reveal an effective therapy for targeted, personalized treatment of aggressive thyroid cancer., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Background: Mucin1 (MUC1), a glycoprotein associated with chemoresistance and an aggressive cancer phenotype, is aberrantly overexpressed in triple-negative breast cancer (TNBC). Recent studies suggest that MUC1 plays a role in modulating cancer cell metabolism and thereby supports tumor growth. Herein, we examined the role of MUC1 in metabolic reprogramming in TNBC., Methods: MUC1 was stably overexpressed in MDA-MB-231 TNBC cells and stably knocked down in MDA-MB-468 cells. We performed liquid chromatography-coupled tandem mass spectrometry-assisted metabolomic analyses and physiological assays, which indicated significant alterations in the metabolism of TNBC cells due to MUC1 expression., Results: Differential analyses identified significant differences in metabolic pathways implicated in cancer cell growth. In particular, MUC1 expression altered glutamine dependency of the cells, which can be attributed in part to the changes in the expression of genes that regulate glutamine metabolism, as observed by real-time PCR analysis. Furthermore, MUC1 expression altered the sensitivity of cells to transaminase inhibitor aminooxyacetate (AOA), potentially by altering glutamine metabolism., Conclusions: Collectively, these results suggest that MUC1 serves as a metabolic regulator in TNBC, facilitating the metabolic reprogramming of glutamine utilization that influences TNBC tumor growth.

Anaplastic thyroid cancer (ATC), one of the most aggressive solid tumors, is characterized by rapid tumor growth and severe metastasis to other organs. Owing to the lack of effective treatment options, ATC has a mortality rate of ∼100% and median survival of less than 5 months. RNAi nanotechnology represents a promising strategy for cancer therapy through nanoparticle (NP) -mediated delivery of RNAi agents (e.g., siRNA) to solid tumors for specific silencing of target genes driving growth and/or metastasis. Nevertheless, the clinical success of RNAi cancer nanotherapies remains elusive in large part because of the suboptimal systemic siRNA NP delivery to tumors and the fact that tumor heterogeneity produces variable NP accumulation and thus, therapeutic response. To address these challenges, we here present an innovative theranostic NP platform composed of a near-infrared (NIR) fluorescent polymer for effective in vivo siRNA delivery to ATC tumors and simultaneous tracking of the tumor accumulation by noninvasive NIR imaging. The NIR polymeric NPs are small (∼50 nm), show long blood circulation and high tumor accumulation, and facilitate tumor imaging. Systemic siRNA delivery using these NPs efficiently silences the expression of V-Raf murine sarcoma viral oncogene homolog B (BRAF) in tumor tissues and significantly suppresses tumor growth and metastasis in an orthotopic mouse model of ATC. These results suggest that this theranostic NP system could become an effective tool for NIR imaging-guided siRNA delivery for personalized treatment of advanced malignancies.

By standard convention, in order to increase the efficacy of metabolite detection from cell culture lysates, metabolite extracts from a large quantity of cells are utilized for multiple reaction monitoring-based metabolomic studies. Metabolomics from a small number of cell extracts offers a potential economical alternative to increased cell numbers, in turn increasing the utility of cell culture-based metabolomics. However, the effect of reduced cell numbers on targeted metabolomic profiling is relatively unstudied. Considering the limited knowledge available of the feasibility and accuracy of microscale cell culture metabolomics, the present study analyzes differences in metabolomic profiles of different cell numbers of three pancreatic cancer cell lines. Specifically, it examines the effects of reduced cell numbers on metabolite profiles by obtaining extracts either directly from microscale culture plates or through serial dilution of increased numbers of cellular metabolite extracts. Our results indicate reduced cell numbers only modestly affect the number of metabolites detected (93% of metabolites detected in cell numbers as low as 104 cells and 97% for 105 cells), independent of the method used to obtain the cells. However, metabolite peak intensities were differentially affected by the reduced cell numbers, with some peak intensities inversely proportional to the cell numbers. To help eliminate such potential inverse relationships, peak intensities for increased cell numbers were excluded from the comparative analysis. Overall, metabolite profiles from microscale culture plates were observed to differ from the serial dilution samples, which may be attributable to the medium-to-cell-number ratios. Finally, findings identify perturbations in metabolomic profiling for cellular extracts from reduced cell numbers, which offer future applications in microscale metabolomic evaluations.

The interaction of programmed cell death-1 and its ligand is widely studied in cancer. Monoclonal antibodies blocking these molecules have had great success but little is known about them in thyroid cancer. We investigated the role of PD-L1 in thyroid cancer with respect to BRAF mutation and MAP kinase pathway activity and the effect of anti PD-L1 antibody therapy on tumor regression and intra-tumoral immune response alone or in combination with BRAF inhibitor (BRAFi). BRAFV600E cells showed significantly higher baseline expression of PD-L1 at mRNA and protein levels compared to BRAFWT cells. MEK inhibitor treatment resulted in a decrease of PD-L1 expression across all cell lines. BRAFi treatment decreased PD-L1 expression in BRAFV600E cells, but paradoxically increased its expression in BRAFWT cells. BRAFV600E mutated patients samples had a higher level of PD-L1 mRNA compared to BRAFWT (p=0.015). Immunocompetent mice (B6129SF1/J) implanted with syngeneic 3747 BRAFV600E/WT P53-/- murine tumor cells were randomized to control, PLX4720, anti PD-L1 antibody and their combination. In this model of aggressive thyroid cancer, control tumor volume reached 782.3±174.6mm3 at two weeks. The combination dramatically reduced tumor volume to 147.3±60.8, compared to PLX4720 (439.3±188.4 mm3, P=0.023) or PD-L1 antibody (716.7±62.1, P<0.001) alone. Immunohistochemistry analysis revealed intense CD8+ CTL infiltration and cytotoxicity and favorable CD8+:Treg ratio compared to each individual treatment. Our results show anti PD-L1 treatment potentiates the effect of BRAFi on tumor regression and intensifies anti tumor immune response in an immunocompetent model of ATC. Clinical trials of this therapeutic combination may be of benefit in patients with ATC.

Background: Investigating BRAF((V600E)) inhibitors (BRAFi) as a strategy to treat patients with aggressive thyroid tumors harboring the BRAF((V600E)) mutant currently is in progress, and drug resistance is expected to pose a challenge. MicroRNAs (miRNAs) are involved in development of resistance to a variety of drugs in different malignancies., Methods: miRNA expression profiles in the human anaplastic thyroid cancer cell line (8505c) were compared with its PLX4720-resistant counterpart (8505c-R) by the use of Illumina deep sequencing. We conducted a functional annotation and pathway analysis of the putative and experimentally validated target genes of the significantly altered miRNAs., Results: We identified 61 known and 2 novel miRNAs whose expression was altered greatly in 8505c-R. Quantitative reverse-transcription polymerase chain reaction validated altered expression of 7 selected miRNAs in 8505c-R and BCPAP-R (PLX4720-resistant papillary thyroid cancer cell line). We found 14 and 25 miRNAs whose expression levels changed substantially in 8505c and 8505c-R, respectively, after treatment with BRAFi. The mitogen-activated protein kinase and phosphatidylinositol 3-kinase-AKT pathways were among the prominent targets of many of the deregulated miRNAs., Conclusion: We have identified a number of miRNAs that could be used as biomarkers of resistance to BRAFi in patients with thyroid cancer. In addition, these miRNAs can be explored as potential therapeutic targets in combination with BRAFi to overcome resistance., (Copyright © 2016 Elsevier Inc. All rights reserved.)

BRAF(V600E) is a common mutation in papillary thyroid carcinoma (PTC) correlated with aggressive features. Our objective was to assess the feasibility and accuracy of a novel RNA-based blood assay to identify individuals with a high-risk tumor mutation in patients with PTC. Patients with benign or malignant thyroid disorders were included between September 2013 and July 2014 before either thyroidectomy (n = 62) or treatment of recurrent or metastatic PTC (n = 8). RNA was isolated from peripheral blood lymphocytes and reverse transcribed and followed by two rounds of nested PCR amplification with a restriction digest specific for wild-type BRAF. BRAF(V600E) levels were quantified with standardization curves. Circulating BRAF(V600E) levels were compared with BRAF mutation status from surgical pathologic DNA-based tissue assays. Testing characteristics and receiving-operator curve using tissue results as the gold standard were assessed. Matched blood and tissue assays for BRAF(V600E) were performed on 70 patients with PTC (stages I to IV, n = 48) or other (n = 22) thyroid tumors. Sixty-three percent of PTC patients tested positive for BRAF(V600E) with conventional tissue assays on surgical specimens. The correlation between the RNA-based blood assay and tissue BRAF status was 0.71. PTC patients harbor detectable BRAF(V600E) circulating tumor cells. This blood assay is feasible and has potential as a biomarker for prognosis, surveillance, clinical decision making, and assessment of treatment response to BRAF-targeted therapies., (Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.)

Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer-related deaths in the US. Cancer-associated cachexia is present in up to 80% of PDAC patients and is associated with aggressive disease and poor prognosis. In the present studies we evaluated an anti-cancer natural product silibinin for its effectiveness in targeting pancreatic cancer aggressiveness and the cachectic properties of pancreatic cancer cells and tumors. Our results demonstrate that silibinin inhibits pancreatic cancer cell growth in a dose-dependent manner and reduces glycolytic activity of cancer cells. Our LC-MS/MS based metabolomics data demonstrates that silibinin treatment induces global metabolic reprogramming in pancreatic cancer cells. Silibinin treatment diminishes c-MYC expression, a key regulator of cancer metabolism. Furthermore, we observed reduced STAT3 signaling in silibinin-treated cancer cells. Overexpression of constitutively active STAT3 was sufficient to substantially revert the silibinin-induced downregulation of c-MYC and the metabolic phenotype. Our in vivo investigations demonstrate that silibinin reduces tumor growth and proliferation in an orthotopic mouse model of pancreatic cancer and prevents the loss of body weight and muscle. It also improves physical activity including grip strength and latency to fall in tumor-bearing mice. In conclusion, silibinin-induced metabolic reprogramming diminishes cell growth and cachectic properties of pancreatic cancer cells and animal models.

MUC16, a transmembrane mucin, facilitates pancreatic adenocarcinoma progression and metastasis. In the current studies, we observed that MUC16 knockdown pancreatic cancer cells exhibit reduced glucose uptake and lactate secretion along with reduced migration and invasion potential, which can be restored by supplementing the culture media with lactate, an end product of aerobic glycolysis. MUC16 knockdown leads to inhibition of mTOR activity and reduced expression of its downstream target c-MYC, a key player in cellular growth, proliferation and metabolism. Ectopic expression of c-MYC in MUC16 knockdown pancreatic cancer cells restores the altered cellular physiology. Our LC-MS/MS based metabolomics studies indicate global metabolic alterations in MUC16 knockdown pancreatic cancer cells, as compared to the controls. Specifically, glycolytic and nucleotide metabolite pools were significantly decreased. We observed similar metabolic alterations that correlated with MUC16 expression in primary tumor tissue specimens from human pancreatic adenocarcinoma cancer patients. Overall, our results demonstrate that MUC16 plays an important role in metabolic reprogramming of pancreatic cancer cells by increasing glycolysis and enhancing motility and invasiveness.

Background: Aberrant energy metabolism is a hallmark of cancer. To fulfill the increased energy requirements, tumor cells secrete cytokines/factors inducing muscle and fat degradation in cancer patients, a condition known as cancer cachexia. It accounts for nearly 20% of all cancer-related deaths. However, the mechanistic basis of cancer cachexia and therapies targeting cancer cachexia thus far remain elusive. A ketogenic diet, a high-fat and low-carbohydrate diet that elevates circulating levels of ketone bodies (i.e., acetoacetate, β-hydroxybutyrate, and acetone), serves as an alternative energy source. It has also been proposed that a ketogenic diet leads to systemic metabolic changes. Keeping in view the significant role of metabolic alterations in cancer, we hypothesized that a ketogenic diet may diminish glycolytic flux in tumor cells to alleviate cachexia syndrome and, hence, may provide an efficient therapeutic strategy., Results: We observed reduced glycolytic flux in tumor cells upon treatment with ketone bodies. Ketone bodies also diminished glutamine uptake, overall ATP content, and survival in multiple pancreatic cancer cell lines, while inducing apoptosis. A decrease in levels of c-Myc, a metabolic master regulator, and its recruitment on glycolytic gene promoters, was in part responsible for the metabolic phenotype in tumor cells. Ketone body-induced intracellular metabolomic reprogramming in pancreatic cancer cells also leads to a significantly diminished cachexia in cell line models. Our mouse orthotopic xenograft models further confirmed the effect of a ketogenic diet in diminishing tumor growth and cachexia., Conclusions: Thus, our studies demonstrate that the cachectic phenotype is in part due to metabolic alterations in tumor cells, which can be reverted by a ketogenic diet, causing reduced tumor growth and inhibition of muscle and body weight loss.

Background: NY-ESO-1 is one of the most immunogenic members of the cancer/testis antigen family and its levels can be increased after exposure to demethylating and deacetylating agents. This cytoplasmic antigen can serve as a potent target for cancer immunotherapy and yet has not been well studied in differentiated thyroid cancer cells., Methods: We studied the baseline expression of NY-ESO-1 messenger RNA and protein before and after exposure to 5-aza-2'-deoxycytidine (DAC) (72 hours) in a panel of thyroid cancer cell lines using quantitative polymerase chain reaction and Western blot. HLA-A2+, NY-ESO-1+ thyroid cell lines were then co-cultured with peripheral blood lymphocytes transduced with NY-ESO-1 specific T-cell receptor (TCR) and assayed for interferon-gamma and Granzyme-B release in the medium. SCID mice injected orthotopically with BCPAP cells were treated with DAC to evaluate for NY-ESO-1 gene expression in vivo., Results: None of the thyroid cancer cell lines showed baseline expression of NY-ESO-1. Three cell lines, BCPAP, TPC-1, and 8505c, showed an increase in NY-ESO-1 gene expression with DAC treatment and were found to be HLA-A2 positive. DAC-treated target BCPAP and TPC-1 tumor cells with up-regulated NY-ESO-1 levels were able to mount an appropriate interferon-gamma and Granzyme-B response upon co-culture with the NY-ESO-1-TCR-transduced peripheral blood lymphocytes. In vivo DAC treatment was able to increase NY-ESO-1 expression in an orthotopic mouse model with BCPAP cells., Conclusion: Our data suggest that many differentiated thyroid cancer cells can be pressed to express immune antigens, which can then be utilized in TCR-based immunotherapeutic interventions.

Anaplastic (ATC) and refractory papillary thyroid cancer (PTC) lack effective treatments. Inhibition of either oncogenic BRAF or SRC has marked anti-tumor effects in mouse models of thyroid cancer, however, neither drug induces notable apoptosis. Here we report that the SRC-inhibitor dasatinib further sensitizes BRAFV600E-positive thyroid cancer cells to the BRAFV600E-inhibitor PLX4720. Combined treatment with PLX4720 and dasatinib synergistically inhibited proliferation and reduced migration in PTC and ATC cells. Whereas PLX4720 did not induce robust apoptosis in thyroid cancer cells, combined treatment with dasatinib induced apoptosis in 4 of 6 lines. In an immunocompetent orthotopic mouse model of ATC, combined PLX4720 and dasatinib treatment significantly reduced tumor volume relative to PLX4720 treatment alone. Immune cell infiltration was increased by PLX4720 treatment and this effect was maintained in mice treated with both PLX4720 and dasatinib. Further, combined treatment significantly increased caspase 3 cleavage in vivo relative to control or either treatment alone. In conclusion, combined PLX4720 and dasatinib treatment induces apoptosis, increases immune cell infiltration and reduces tumor volume in a preclinical model of ATC, suggesting that the combination of these FDA-approved drugs may have potential for the treatment of patients with ATC or refractory PTC.

Background: While the development of new treatments for aggressive thyroid cancer has advanced in the last 10 years, progress has trailed headways made with other malignancies. A lack of reliable authenticated human cell lines and reproducible animal models is one major roadblock to preclinical testing of novel therapeutics. Existing xenograft and orthotopic mouse models of aggressive thyroid cancer rely on the implantation of highly passaged human thyroid carcinoma lines in immunodeficient mice. Genetically engineered models of papillary and undifferentiated (anaplastic) thyroid carcinoma (PTC and ATC) are immunocompetent; however, slow and stochastic tumor development hinders high-throughput testing. Novel models of PTC and ATC in which tumors arise rapidly and synchronously in immunocompetent mice would facilitate the investigation of novel therapeutics and approaches., Methods: We characterized and utilized mouse cell lines derived from PTC and ATC tumors arising in genetically engineered mice with thyroid-specific expression of endogenous Braf(V600E/WT) and deletion of either Trp53 (p53) or Pten. These murine thyroid cancer cells were transduced with luciferase- and GFP-expressing lentivirus and implanted into the thyroid glands of immunocompetent syngeneic B6129SF1/J mice in which the growth characteristics were assessed., Results: Large locally aggressive thyroid tumors form within one week of implantation. Tumors recapitulate their histologic subtype, including well-differentiated PTC and ATC, and exhibit CD3+, CD8+, B220+, and CD163+ immune cell infiltration. Tumor progression can be followed in vivo using luciferase and ex vivo using GFP. Metastatic spread is not detected at early time points., Conclusions: We describe the development of the next generation of murine orthotopic thyroid cancer models. The implantation of genetically defined murine BRAF-mutated PTC and ATC cell lines into syngeneic mice results in rapid and synchronous tumor formation. This model allows for preclinical investigation of novel therapeutics and/or therapeutic combinations in the context of a functional immune system.