TNFα is a key mediator of immune, chemotherapy and radiotherapy-induced cytotoxicity, but several cancers, including head and neck squamous cell carcinomas (HNSCC), display resistance to TNFα due to ...activation of the canonical NFκB pro-survival pathway. However, direct targeting of this pathway is associated with significant toxicity; thus, it is vital to identify novel mechanism(s) contributing to NFκB activation and TNFα resistance in cancer cells. Here, we demonstrate that the expression of proteasome-associated deubiquitinase USP14 is significantly increased in HNSCC and correlates with worse progression free survival in Human Papillomavirus (HPV)- HNSCC. Inhibition or depletion of USP14 inhibited the proliferation and survival of HNSCC cells. Further, USP14 inhibition reduced both basal and TNFα-inducible NFκB activity, NFκB-dependent gene expression and the nuclear translocation of the NFκB subunit RELA. Mechanistically, USP14 bound to both RELA and IκBα and reduced IκBα K48-ubiquitination leading to the degradation of IκBα, a critical inhibitor of the canonical NFκB pathway. Furthermore, we demonstrated that b-AP15, an inhibitor of USP14 and UCHL5, sensitized HNSCC cells to TNFα-mediated cell death, as well as radiation-induced cell death in vitro. Finally, b-AP15 delayed tumor growth and enhanced survival, both as a monotherapy and in combination with radiation, in HNSCC tumor xenograft models in vivo, which could be significantly attenuated by TNFα depletion. These data offer new insights into the activation of NFκB signaling in HNSCC and demonstrate that small molecule inhibitors targeting the ubiquitin pathway warrant further investigation as a novel therapeutic avenue to sensitize these cancers to TNFα- and radiation-induced cytotoxicity.
Patients with germline fumarate hydratase (FH) mutation are predisposed to develop aggressive kidney cancer with few treatment options and poor therapeutic outcomes. Activity of the proto-oncogene ...ABL1 is upregulated in FH-deficient kidney tumors and drives a metabolic and survival signaling network necessary to cope with impaired mitochondrial function and abnormal accumulation of intracellular fumarate. Excess fumarate indirectly stimulates ABL1 activity, while restoration of wild-type FH abrogates both ABL1 activation and the cytotoxicity caused by ABL1 inhibition or knockdown. ABL1 upregulates aerobic glycolysis via the mTOR/HIF1α pathway and neutralizes fumarate-induced proteotoxic stress by promoting nuclear localization of the antioxidant response transcription factor NRF2. Our findings identify ABL1 as a pharmacologically tractable therapeutic target in glycolytically dependent, oxidatively stressed tumors.
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•Elevated intracellular fumarate indirectly activates ABL1•ABL1 promotes HIF-dependent aerobic glycolysis in FH-deficient tumor cells•ABL1 stimulates the NRF2-dependent antioxidant response pathway•ABL1 inhibition provides a strategy to treat oxidatively stressed tumors
Sourbier et al. report ABL1 activation in fumarate hydratase (FH)-deficient kidney tumors, promoting aerobic glycolysis and NRF2-mediated antioxidant defense. ABL1 inhibition may be clinically useful for the treatment of FH-deficient kidney cancer and other glycolytic, oxidatively stressed tumors.
In hypoxic tumor microenvironments, the strongly reducing redox environment reduces evofosfamide (TH-302) to release a cytotoxic bromo-isophosphoramide (Br-IPM) moiety. This drug therefore ...preferentially attacks hypoxic regions in tumors where other standard anticancer treatments such as chemotherapy and radiation therapy are often ineffective. Various combination therapies with evofosfamide have been proposed and tested in preclinical and clinical settings. However, the treatment effect of evofosfamide monotherapy on tumor hypoxia has not been fully understood, partly due to the lack of quantitative methods to assess tumor pO
. Here, we use quantitative pO
imaging by electron paramagnetic resonance (EPR) to evaluate the change in tumor hypoxia in response to evofosfamide treatment using two pancreatic ductal adenocarcinoma xenograft models: MIA Paca-2 tumors responding to evofosfamide and Su.86.86 tumors that do not respond.
EPR imaging showed that oxygenation improved globally after evofosfamide treatment in hypoxic MIA Paca-2 tumors, in agreement with the
results obtained from hypoxia staining by pimonidazole and in apparent contrast to the decrease in K
observed in dynamic contrast-enhanced magnetic resonance imaging (DCE MRI).
The observation that evofosfamide not only kills the hypoxic region of the tumor but also improves oxygenation in the residual tumor regions provides a rationale for combination therapies using radiation and antiproliferatives post evofosfamide for improved outcomes.
This study suggests that reoxygenation after evofosfamide treatment is due to decreased oxygen demand rather than improved perfusion. Following the change in pO
after treatment may therefore yield a way of monitoring treatment response.
. 35, 904-915.
No conventional therapy exists for salivary hypofunction in surviving head and neck cancer patients with Radiation Therapy Oncology Group late grade 2–3 toxicity. We conducted a phase I clinical ...trial to test the safety and biologic efficacy of serotype 5, adenoviral-mediated aquaporin-1 cDNA transfer to a single previously irradiated parotid gland in 11 subjects using an open label, single-dose, dose-escalation design (AdhAQP1 vector; four dose tiers from 4.8 × 10 ⁷ to 5.8 × 10 ⁹ vector particles per gland). Treated subjects were followed at scheduled intervals. Multiple safety parameters were measured and biologic efficacy was evaluated with measurements of parotid salivary flow rate. Symptoms were assessed with a visual analog scale. All subjects tolerated vector delivery and study procedures well over the 42-d study period reported. No deaths, serious adverse events, or dose-limiting toxicities occurred. Generally, few adverse events occurred, and all were considered mild or moderate. No consistent changes were found in any clinical chemistry and hematology parameters measured. Objective responses were seen in six subjects, all at doses <5.8 × 10 ⁹ vector particles per gland. Five of these six subjects also experienced subjective improvement in xerostomia. AdhAQP1 vector delivery to a single parotid gland was safe and transfer of the hAQP1 cDNA increased parotid flow and relieved symptoms in a subset of subjects.
The presence of structural water in tungsten oxides leads to a transition in the energy storage mechanism from battery-type intercalation (limited by solid state diffusion) to pseudocapacitance ...(limited by surface kinetics). Here, we demonstrate that these electrochemical mechanisms are linked to the mechanical response of the materials during intercalation of protons and present a pathway to utilize the mechanical coupling for local studies of electrochemistry. Operando atomic force microscopy dilatometry is used to measure the deformation of redox-active energy storage materials and to link the local nanoscale deformation to the electrochemical redox process. This technique reveals that the local mechanical deformation of the hydrated tungsten oxide is smaller and more gradual than the anhydrous oxide and occurs without hysteresis during the intercalation and deintercalation processes. The ability of layered materials with confined structural water to minimize mechanical deformation likely contributes to their fast energy storage kinetics.
Radiotherapy is highly effective due to its ability to physically focus the treatment to target the tumor while sparing normal tissue and its ability to be combined with systemic therapy. This ...systemic therapy can be utilized before radiotherapy as an adjuvant or induction treatment, during radiotherapy as a radiation "sensitizer," or following radiotherapy as a part of combined modality therapy. As part of a unique concept of using radiation as "focused biology," we investigated how tumors and normal tissues adapt to clinically relevant multifraction (MF) and single-dose (SD) radiation to observe whether the adaptations can induce susceptibility to cell killing by available drugs or by immune enhancement. We identified an adaptation occurring after MF (3 × 2 Gy) that induced cell killing when AKT-mTOR inhibitors were delivered following cessation of radiotherapy. In addition, we identified inducible changes in integrin expression 2 months following cessation of radiotherapy that differ between MF (1 Gy × 10) and SD (10 Gy) that remain targetable compared with preradiotherapy. Adaptation is reflected across different "omics" studies, and thus the range of possible molecular targets is not only broad but also time, dose, and schedule dependent. While much remains to be studied about the radiation adaptive response, radiation should be characterized by its molecular perturbations in addition to physical dose. Consideration of the adaptive effects should result in the design of a tailored radiotherapy treatment plan that accounts for specific molecular changes to be targeted as part of precision multimodality cancer treatment.
Structural equation modeling (SEM) is increasingly being chosen by researchers as a framework for gaining scientific insights from the quantitative analyses of data. New ideas and methods emerging ...from the study of causality, influences from the field of graphical modeling, and advances in statistics are expanding the rigor, capability, and even purpose of SEM. Guidelines for implementing the expanded capabilities of SEM are currently lacking. In this paper we describe new developments in SEM that we believe constitute a third-generation of the methodology. Most characteristic of this new approach is the generalization of the structural equation model as a causal graph. In this generalization, analyses are based on graph theoretic principles rather than analyses of matrices. Also, new devices such as metamodels and causal diagrams, as well as an increased emphasis on queries and probabilistic reasoning, are now included. Estimation under a graph theory framework permits the use of Bayesian or likelihood methods. The guidelines presented start from a declaration of the goals of the analysis. We then discuss how theory frames the modeling process, requirements for causal interpretation, model specification choices, selection of estimation method, model evaluation options, and use of queries, both to summarize retrospective results and for prospective analyses.
The illustrative example presented involves monitoring data from wetlands on Mount Desert Island, home of Acadia National Park. Our presentation walks through the decision process involved in developing and evaluating models, as well as drawing inferences from the resulting prediction equations. In addition to evaluating hypotheses about the connections between human activities and biotic responses, we illustrate how the structural equation (SE) model can be queried to understand how interventions might take advantage of an environmental threshold to limit
Typha
invasions.
The guidelines presented provide for an updated definition of the SEM process that subsumes the historical matrix approach under a graph-theory implementation. The implementation is also designed to permit complex specifications and to be compatible with various estimation methods. Finally, they are meant to foster the use of probabilistic reasoning in both retrospective and prospective considerations of the quantitative implications of the results.
Nitroxide free radicals can serve as redox-sensitive MRI contrast agents useful to image the redox status of tissue of interest. In this study, the effect of oxygen content in the inspired gas on the ...kinetics of metabolism of three nitroxides has been evaluated in the muscle and tumor in mice.
SCC tumors (approximate size of 1.0 cm3) on the right hind leg of female C3H/Hen MTV- mice were prepared. Three nitroxides, 3-carboxy-2,2,5,5-tetramethylpyrrolidine-N-oxyl (CxP), 3-carbamoyl-2,2,5,5-tetramethylpyrrolidine-N-oxyl (CmP), and 4-hydroxy-tetramethylpiperidine-N-oxyl (TEMPOL), having different lipophilicities were compared using MR redox imaging. T1-mapping of the tissues was obtained using a multi-slice multi-echo (MSME) sequence with several TRs.
The three nitroxides showed differences in accumulation and metabolism/clearance in muscle and tumor. The cell impermeable nitroxide CxP displayed kinetic patterns of slow enhancement followed by a slow decline typical of clearance rather than metabolism. The cell permeable CmP on the other hand showed a relatively faster uptake and metabolism with a modestly higher rate of metabolism in the tumor than muscle. The TEMPOL on the other hand displayed a rapid uptake and reduction with a trend of significantly rapid decay rate in tumor tissue, while slightly higher maximum signal intensity and slower decay rate was observed in normal muscle. The reduction rate of TEMPOL in the tumor was significantly enhanced when the breathing gas had 100%-oxygen while it was not significantly different in the muscle. EPR oximetry studies monitoring the oxygen dependent linewidth of TEMPOL showed that the pO2 in the healthy tissue during carbogen breathing significantly increased normal tissue pO2 compared to air breathing whereas breathing 100%-oxygen made normal tissue slight hypoxic. Since TEMPOL is a radioprotector, our studies show that a combination of 100%-oxygen breathing and TEMPOL has a potential to enhance radioprotective effects to normal tissue.
•100%-O2 breathing makes the decay rate of TEMPOL in tumor tissue faster.•100%-O2 breathing makes the decay rate of TEMPOL in the normal muscle slower.•100%-O2 breathing did not affect to the normal tissue pO2.•100%-O2 makes difference of TEMPOL conc. between normal and tumor tissues larger.
To evaluate the potential of hyperpolarized 1-
C-pyruvate magnetic resonance spectroscopic imaging (MRSI) of prostate cancer as a predictive biomarker for targeting the Warburg effect.
Two human ...prostate cancer cell lines (DU145 and PC3) were grown as xenografts. The conversion of pyruvate to lactate in xenografts was measured with hyperpolarized 1-
C-pyruvate MRSI after systemic delivery of 1-
C pyruvic acid. Steady-state metabolomic analysis of xenograft tumors was performed with mass spectrometry and steady-state lactate concentrations were measured with proton (
H) MRS. Perfusion and oxygenation of xenografts were measured with electron paramagnetic resonance (EPR) imaging with OX063. Tumor growth was assessed after lactate dehydrogenase (LDH) inhibition with FX-11 (42 μg/mouse/day for 5 days × 2 weekly cycles). Lactate production, pyruvate uptake, extracellular acidification rates, and oxygen consumption of the prostate cancer cell lines were analyzed
LDH activity was assessed in tumor homogenates.
DU145 tumors demonstrated an enhanced conversion of pyruvate to lactate with hyperpolarized 1-
C-pyruvate MRSI compared with PC3 and a corresponding greater sensitivity to LDH inhibition. No difference was observed between PC3 and DU145 xenografts in steady-state measures of pyruvate fermentation, oxygenation, or perfusion. The two cell lines exhibited similar sensitivity to FX-11
LDH activity correlated to FX-11 sensitivity.
Hyperpolarized 1-
C-pyruvate MRSI of prostate cancer predicts efficacy of targeting the Warburg effect.
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