Publisher Correction: In vivo imaging of mitochondrial membrane potential in non-small-cell lung cancer.

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

In vivo imaging of mitochondrial membrane potential in non-small-cell lung cancer.

Mitochondria are essential regulators of cellular energy and metabolism, and have a crucial role in sustaining the growth and survival of cancer cells. A central function of mitochondria is the synthesis of ATP by oxidative phosphorylation, known as mitochondrial bioenergetics. Mitochondria maintain oxidative phosphorylation by creating a membrane potential gradient that is generated by the electron transport chain to drive the synthesis of ATP1. Mitochondria are essential for tumour initiation and maintaining tumour cell growth in cell culture and xenografts2,3. However, our understanding of oxidative mitochondrial metabolism in cancer is limited because most studies have been performed in vitro in cell culture models. This highlights a need for in vivo studies to better understand how oxidative metabolism supports tumour growth. Here we measure mitochondrial membrane potential in non-small-cell lung cancer in vivo using a voltage-sensitive, positron emission tomography (PET) radiotracer known as 4-[18F]fluorobenzyl-triphenylphosphonium (18F-BnTP)4. By using PET imaging of 18F-BnTP, we profile mitochondrial membrane potential in autochthonous mouse models of lung cancer, and find distinct functional mitochondrial heterogeneity within subtypes of lung tumours. The use of 18F-BnTP PET imaging enabled us to functionally profile mitochondrial membrane potential in live tumours.

Individual cristae within the same mitochondrion display different membrane potentials and are functionally independent.

The mitochondrial membrane potential (ΔΨm ) is the main driver of oxidative phosphorylation (OXPHOS). The inner mitochondrial membrane (IMM), consisting of cristae and inner boundary membranes (IBM), is considered to carry a uniform ΔΨm . However, sequestration of OXPHOS components in cristae membranes necessitates a re-examination of the equipotential representation of the IMM. We developed an approach to monitor ΔΨm at the resolution of individual cristae. We found that the IMM was divided into segments with distinct ΔΨm , corresponding to cristae and IBM. ΔΨm was higher at cristae compared to IBM. Treatment with oligomycin increased, whereas FCCP decreased, ΔΨm heterogeneity along the IMM. Impairment of cristae structure through deletion of MICOS-complex components or Opa1 diminished this intramitochondrial heterogeneity of ΔΨm . Lastly, we determined that different cristae within the individual mitochondrion can have disparate membrane potentials and that interventions causing acute depolarization may affect some cristae while sparing others. Altogether, our data support a new model in which cristae within the same mitochondrion behave as independent bioenergetic units, preventing the failure of specific cristae from spreading dysfunction to the rest.

Targeted inhibition of gut bacterial ß-glucuronidase activity enhances anticancer drug efficacy.

Irinotecan treats a range of solid tumors, but its effectiveness is severely limited by gastrointestinal (GI) tract toxicity caused by gut bacterial ß-glucuronidase (GUS) enzymes. Targeted bacterial GUS inhibitors have been shown to partially alleviate irinotecan-induced GI tract damage and resultant diarrhea in mice. Here, we unravel the mechanistic basis for GI protection by gut microbial GUS inhibitors using in vivo models. We use in vitro, in fimo, and in vivo models to determine whether GUS inhibition alters the anticancer efficacy of irinotecan. We demonstrate that a single dose of irinotecan increases GI bacterial GUS activity in 1 d and reduces intestinal epithelial cell proliferation in 5 d, both blocked by a single dose of a GUS inhibitor. In a tumor xenograft model, GUS inhibition prevents intestinal toxicity and maintains the antitumor efficacy of irinotecan. Remarkably, GUS inhibitor also effectively blocks the striking irinotecan-induced bloom of Enterobacteriaceae in immune-deficient mice. In a genetically engineered mouse model of cancer, GUS inhibition alleviates gut damage, improves survival, and does not alter gut microbial composition; however, by allowing dose intensification, it dramatically improves irinotecan's effectiveness, reducing tumors to a fraction of that achieved by irinotecan alone, while simultaneously promoting epithelial regeneration. These results indicate that targeted gut microbial enzyme inhibitors can improve cancer chemotherapeutic outcomes by protecting the gut epithelium from microbial dysbiosis and proliferative crypt damage.

Entinostat induces antitumor immune responses through immune editing of tumor neoantigens.

Although immune-checkpoint inhibitors (ICIs) have been a remarkable advancement in bladder cancer treatment, the response rate to single-agent ICIs remains suboptimal. There has been substantial interest in the use of epigenetic agents to enhance ICI efficacy, although precisely how these agents potentiate ICI response has not been fully elucidated. We identified entinostat, a selective HDAC1/3 inhibitor, as a potent antitumor agent in our immune-competent bladder cancer mouse models (BBN963 and BBN966). We demonstrate that entinostat selectively promoted immune editing of tumor neoantigens, effectively remodeling the tumor immune microenvironment, resulting in a robust antitumor response that was cell autonomous, dependent upon antigen presentation, and associated with increased numbers of neoantigen-specific T cells. Finally, combination treatment with anti-PD-1 and entinostat led to complete responses and conferred long-term immunologic memory. Our work defines a tumor cell-autonomous mechanism of action for entinostat and a strong preclinical rationale for the combined use of entinostat and PD-1 blockade in bladder cancer.

An Automated Multidose Synthesis of the Potentiometric PET Probe 4-[18F]Fluorobenzyl-Triphenylphosphonium ([18F]FBnTP).

PURPOSE: The aim of this study was the automated synthesis of the mitochondrial membrane potential sensor 4-[18F]fluorobenzyl-triphenylphosphonium ([18F]FBnTP) on a commercially available synthesizer in activity yields (AY) that allow for imaging of multiple patients. PROCEDURES: A three-pot, four-step synthesis was implemented on the ELIXYS FLEX/CHEM radiosynthesizer (Sofie Biosciences) and optimized for radiochemical yield (RCY), radiochemical purity (RCP) as well as chemical purity during several production runs (n = 24). The compound was purified by solid-phase extraction (SPE) with a Sep-Pak Plus Accell CM cartridge, thereby avoiding HPLC purification. RESULTS: Under optimized conditions, AY of 1.4-2.2 GBq of [18F]FBnTP were obtained from 9.4 to 12.0 GBq [18F]fluoride in 90-92 min (RCY = 28.6 ± 5.1 % with n = 3). Molar activities ranged from 80 to 99 GBq/µmol at the end of synthesis. RCP of final formulations was > 99 % at the end of synthesis and > 95 % after 8 h. With starting activities of 23.2-33.0 GBq, RCY decreased to 16.1 ± 0.4 % (n = 3). The main cause of the decline in RCY when high amounts of [18F]fluoride are used is radiolytic decomposition of [18F]FBnTP during SPE purification. CONCLUSIONS: In initial attempts, the probe was synthesized with RCY < 0.6 % when starting activities up to 44.6 GBq were used. Rapid radiolysis of the intermediate 4-[18F]fluorobenzaldehyde and the final product [18F]FBnTP during purification was identified as the main cause for low yields in high-activity runs. Radiolytic decomposition was hindered by the addition of radical scavengers during synthesis, purification, and formulation, thereby improving AY and RCP. The formulated probe in injectable form was synthesized without the use of HPLC and passed all applicable quality control tests.

Utilizing 18F-FDG PET/CT Imaging and Quantitative Histology to Measure Dynamic Changes in the Glucose Metabolism in Mouse Models of Lung Cancer.

A hallmark of advanced tumors is a switch to aerobic glycolysis that is readily measured by [18F]-2-fluoro-2-deoxy-D-glucose positron emission tomography (18F-FDG PET) imaging. Co-mutations in the KRAS proto-oncogene and the LKB1 tumor suppressor gene are frequent events in lung cancer that drive hypermetabolic, glycolytic tumor growth. A critical pathway regulating the growth and metabolism of these tumors is the mechanistic target of the rapamycin (mTOR) pathway, which can be effectively targeted using selective catalytic mTOR kinase inhibitors. The mTOR inhibitor MLN0128 suppresses glycolysis in mice bearing tumors with Kras and Lkb1 co-mutations, referred to as KL mice. The therapy response in KL mice is first measured by 18F-FDG PET and computed tomography (CT) imaging before and after the delivery of MLN0128. By utilizing 18F-FDG PET/CT, researchers are able to measure dynamic changes in the glucose metabolism in genetically engineered mouse models (GEMMs) of lung cancer following a therapeutic intervention with targeted therapies. This is followed by ex vivo autoradiography and a quantitative immunohistochemical (qIHC) analysis using morphometric software. The use of qIHC enables the detection and quantification of distinct changes in the biomarker profiles following treatment as well as the characterization of distinct tumor pathologies. The coupling of PET imaging to quantitative histology is an effective strategy to identify metabolic and therapeutic responses in vivo in mouse models of disease.

Sodium-glucose transporter 2 is a diagnostic and therapeutic target for early-stage lung adenocarcinoma.

The diagnostic definition of indeterminate lung nodules as malignant or benign poses a major challenge for clinicians. We discovered a potential marker, the sodium-dependent glucose transporter 2 (SGLT2), whose activity identified metabolically active lung premalignancy and early-stage lung adenocarcinoma (LADC). We found that SGLT2 is expressed early in lung tumorigenesis and is found specifically in premalignant lesions and well-differentiated adenocarcinomas. SGLT2 activity could be detected in vivo by positron emission tomography (PET) with the tracer methyl 4-deoxy-4-[18F] fluoro-alpha-d-glucopyranoside (Me4FDG), which specifically detects SGLT activity. Using a combination of immunohistochemistry and Me4FDG PET, we identified high expression and functional activity of SGLT2 in lung premalignancy and early-stage/low-grade LADC. Furthermore, selective targeting of SGLT2 with FDA-approved small-molecule inhibitors, the gliflozins, greatly reduced tumor growth and prolonged survival in autochthonous mouse models and patient-derived xenografts of LADC. Targeting SGLT2 in lung tumors may intercept lung cancer progression at early stages of development by pairing Me4FDG PET imaging with therapy using SGLT2 inhibitors.

The GSK3 Signaling Axis Regulates Adaptive Glutamine Metabolism in Lung Squamous Cell Carcinoma.

Altered metabolism is a hallmark of cancer growth, forming the conceptual basis for development of metabolic therapies as cancer treatments. We performed in vivo metabolic profiling and molecular analysis of lung squamous cell carcinoma (SCC) to identify metabolic nodes for therapeutic targeting. Lung SCCs adapt to chronic mTOR inhibition and suppression of glycolysis through the GSK3α/ß signaling pathway, which upregulates glutaminolysis. Phospho-GSK3α/ß protein levels are predictive of response to single-therapy mTOR inhibition while combinatorial treatment with the glutaminase inhibitor CB-839 effectively overcomes therapy resistance. In addition, we identified a conserved metabolic signature in a broad spectrum of hypermetabolic human tumors that may be predictive of patient outcome and response to combined metabolic therapies targeting mTOR and glutaminase.

MYC activation cooperates with Vhl and Ink4a/Arf loss to induce clear cell renal cell carcinoma.

Renal carcinoma is a common and aggressive malignancy whose histopathogenesis is incompletely understood and that is largely resistant to cytotoxic chemotherapy. We present two mouse models of kidney cancer that recapitulate the genomic alterations found in human papillary (pRCC) and clear cell RCC (ccRCC), the most common RCC subtypes. MYC activation results in highly penetrant pRCC tumours (MYC), while MYC activation, when combined with Vhl and Cdkn2a (Ink4a/Arf) deletion (VIM), produce kidney tumours that approximate human ccRCC. RNAseq of the mouse tumours demonstrate that MYC tumours resemble Type 2 pRCC, which are known to harbour MYC activation. Furthermore, VIM tumours more closely simulate human ccRCC. Based on their high penetrance, short latency, and histologic fidelity, these models of papillary and clear cell RCC should be significant contributions to the field of kidney cancer research.

Targeted Inhibition of EGFR and Glutaminase Induces Metabolic Crisis in EGFR Mutant Lung Cancer.

Cancer cells exhibit increased use of nutrients, including glucose and glutamine, to support the bioenergetic and biosynthetic demands of proliferation. We tested the small-molecule inhibitor of glutaminase CB-839 in combination with erlotinib on epidermal growth factor receptor (EGFR) mutant non-small cell lung cancer (NSCLC) as a therapeutic strategy to simultaneously impair cancer glucose and glutamine utilization and thereby suppress tumor growth. Here, we show that CB-839 cooperates with erlotinib to drive energetic stress and activate the AMP-activated protein kinase (AMPK) pathway in EGFR (del19) lung tumors. Tumor cells undergo metabolic crisis and cell death, resulting in rapid tumor regression in vivo in mouse NSCLC xenografts. Consistently, positron emission tomography (PET) imaging with 18F-fluoro-2-deoxyglucose (18F-FDG) and 11C-glutamine (11C-Gln) of xenografts indicated reduced glucose and glutamine uptake in tumors following treatment with CB-839 + erlotinib. Therefore, PET imaging with 18F-FDG and 11C-Gln tracers can be used to non-invasively measure metabolic response to CB-839 and erlotinib combination therapy.

mTOR inhibition induces compensatory, therapeutically targetable MEK activation in renal cell carcinoma.

Rapamycin derivatives allosterically targeting mTOR are currently FDA approved to treat advanced renal cell carcinoma (RCC), and catalytic inhibitors of mTOR/PI3K are now in clinical trials for treating various solid tumors. We sought to investigate the relative efficacy of allosteric versus catalytic mTOR inhibition, evaluate the crosstalk between the mTOR and MEK/ERK pathways, as well as the therapeutic potential of dual mTOR and MEK inhibition in RCC. Pharmacologic (rapamycin and BEZ235) and genetic manipulation of the mTOR pathway were evaluated by in vitro assays as monotherapy as well as in combination with MEK inhibition (GSK1120212). Catalytic mTOR inhibition with BEZ235 decreased proliferation and increased apoptosis better than allosteric mTOR inhibition with rapamycin. While mTOR inhibition upregulated MEK/ERK signaling, concurrent inhibition of both pathways had enhanced therapeutic efficacy. Finally, primary RCC tumors could be classified into subgroups [(I) MEK activated, (II) Dual MEK and mTOR activated, (III) Not activated, and (IV) mTOR activated] based on their relative activation of the PI3K/mTOR and MEK pathways. Patients with mTOR only activated tumors had the worst prognosis. In summary, dual targeting of the mTOR and MEK pathways in RCC can enhance therapeutic efficacy and primary RCC can be subclassified based on their relative levels of mTOR and MEK activation with potential therapeutic implications.

Erythropoietin promotes breast tumorigenesis through tumor-initiating cell self-renewal.

Erythropoietin (EPO) is a hormone that induces red blood cell production. In its recombinant form, EPO is the one of most prescribed drugs to treat anemia, including that arising in cancer patients. In randomized trials, EPO administration to cancer patients has been associated with decreased survival. Here, we investigated the impact of EPO modulation on tumorigenesis. Using genetically engineered mouse models of breast cancer, we found that EPO promoted tumorigenesis by activating JAK/STAT signaling in breast tumor-initiating cells (TICs) and promoted TIC self renewal. We determined that EPO was induced by hypoxia in breast cancer cell lines, but not in human mammary epithelial cells. Additionally, we demonstrated that high levels of endogenous EPO gene expression correlated with shortened relapse-free survival and that pharmacologic JAK2 inhibition was synergistic with chemotherapy for tumor growth inhibition in vivo. These data define an active role for endogenous EPO in breast cancer progression and breast TIC self-renewal and reveal a potential application of EPO pathway inhibition in breast cancer therapy.

HIF1α and HIF2α independently activate SRC to promote melanoma metastases.

Malignant melanoma is characterized by a propensity for early lymphatic and hematogenous spread. The hypoxia-inducible factor (HIF) family of transcription factors is upregulated in melanoma by key oncogenic drivers. HIFs promote the activation of genes involved in cancer initiation, progression, and metastases. Hypoxia has been shown to enhance the invasiveness and metastatic potential of tumor cells by regulating the genes involved in the breakdown of the ECM as well as genes that control motility and adhesion of tumor cells. Using a Pten-deficient, Braf-mutant genetically engineered mouse model of melanoma, we demonstrated that inactivation of HIF1α or HIF2α abrogates metastasis without affecting primary tumor formation. HIF1α and HIF2α drive melanoma invasion and invadopodia formation through PDGFRα and focal adhesion kinase-mediated (FAK-mediated) activation of SRC and by coordinating ECM degradation via MT1-MMP and MMP2 expression. These results establish the importance of HIFs in melanoma progression and demonstrate that HIF1α and HIF2α activate independent transcriptional programs that promote metastasis by coordinately regulating cell invasion and ECM remodeling.

Metabolic and functional genomic studies identify deoxythymidylate kinase as a target in LKB1-mutant lung cancer.

The LKB1/STK11 tumor suppressor encodes a serine/threonine kinase, which coordinates cell growth, polarity, motility, and metabolism. In non-small cell lung carcinoma, LKB1 is somatically inactivated in 25% to 30% of cases, often concurrently with activating KRAS mutations. Here, we used an integrative approach to define novel therapeutic targets in KRAS-driven LKB1-mutant lung cancers. High-throughput RNA interference screens in lung cancer cell lines from genetically engineered mouse models driven by activated KRAS with or without coincident Lkb1 deletion led to the identification of Dtymk, encoding deoxythymidylate kinase (DTYMK), which catalyzes dTTP biosynthesis, as synthetically lethal with Lkb1 deficiency in mouse and human lung cancer lines. Global metabolite profiling showed that Lkb1-null cells had a striking decrease in multiple nucleotide metabolites as compared with the Lkb1-wild-type cells. Thus, LKB1-mutant lung cancers have deficits in nucleotide metabolism that confer hypersensitivity to DTYMK inhibition, suggesting that DTYMK is a potential therapeutic target in this aggressive subset of tumors.

State of the science: an update on renal cell carcinoma.

Renal cell carcinomas (RCC) are emerging as a complex set of diseases that are having a major socioeconomic impact and showing a continued rise in incidence throughout the world. As the field of urologic oncology faces these trends, several major genomic and mechanistic discoveries are altering our core understanding of this multitude of cancers, including several new rare subtypes of renal cancers. In this review, these new findings are examined and placed in the context of the well-established association of clear cell RCC (ccRCC) with mutations in the von Hippel-Lindau (VHL) gene and resultant aberrant hypoxia inducible factor (HIF) signaling. The impact of novel ccRCC-associated genetic lesions on chromatin remodeling and epigenetic regulation is explored. The effects of VHL mutation on primary ciliary function, extracellular matrix homeostasis, and tumor metabolism are discussed. Studies of VHL proteostasis, with the goal of harnessing the proteostatic machinery to refunctionalize mutant VHL, are reviewed. Translational efforts using molecular tools to elucidate discriminating features of ccRCC tumors and develop improved prognostic and predictive algorithms are presented, and new therapeutics arising from the earliest molecular discoveries in ccRCC are summarized. By creating an integrated review of the key genomic and molecular biological disease characteristics of ccRCC and placing these data in the context of the evolving therapeutic landscape, we intend to facilitate interaction among basic, translational, and clinical researchers involved in the treatment of this devastating disease, and accelerate progress toward its ultimate eradication.

Leptin stimulates pituitary prolactin release through an extracellular signal-regulated kinase-dependent pathway.

Leptin was initially identified as a regulator of appetite and weight control centers in the hypothalamus, but appears to be involved in a number of physiological processes. This study was carried out to examine the possible role of leptin in regulating prolactin (PRL) release using the teleost pituitary model system. This advantageous system allows isolation of a nearly pure population of lactotropes in their natural, in situ aggregated state. The rostral pars distalis were dissected from tilapia pituitaries and exposed to varying concentrations of leptin (0, 1, 10, 100 nM) for 1 h. Release of PRL was stimulated by leptin in a potent and concentration-dependent manner. A time-course experiment showed that the strongest response in PRL release with leptin occurs within the first hour (approximately sixfold), and stimulation was sustained after 16 h (approximately twofold). Many of the actions of leptin are mediated by the activation of extracellular signal-regulated kinase (ERK1/2) but nothing is known about the cellular mechanisms by which leptin might regulate PRL secretion in vertebrates. We therefore tested whether ERK1/2 might be involved in the leptin PRL response and found that the ERK inhibitor, PD98059, hindered leptin-induced PRL release. We further analyzed leptin response by quantifying tyrosine and threonine phosphorylation of ERK1/2 using western blots. One hour incubation with leptin induced a concentration-dependent increase in phosphorylated, and thus active, ERK1/2. Our data show that leptin is a powerful stimulator of in vitro PRL release and that its actions occur in part through stimulation of ERK1/2.