Here, we interrogated head and neck cancer (HNSCC) specimens (n = 12) to examine if different metabolic compartments (oxidative vs. glycolytic) co-exist in human tumors. A large panel of well-established biomarkers was employed to determine the metabolic state of proliferative cancer cells. Interestingly, cell proliferation in cancer cells, as marked by Ki-67 immunostaining, was strictly correlated with oxidative mitochondrial metabolism (OXPHOS) and the uptake of mitochondrial fuels, as detected via MCT1 expression (p < 0.001). More specifically, three metabolic tumor compartments were delineated: (1) proliferative and mitochondrial-rich cancer cells (Ki-67+/TOMM20+/COX+/MCT1+); (2) non-proliferative and mitochondrial-poor cancer cells (Ki-67−/TOMM20−/COX−/MCT1−); and (3) non-proliferative and mitochondrial-poor stromal cells (Ki-67−/TOMM20−/COX−/MCT1−). In addition, high oxidative stress (MCT4+) was very specific for cancer tissues. Thus, we next evaluated the prognostic value of MCT4 in a second independent patient cohort (n = 40). Most importantly, oxidative stress (MCT4+) in non-proliferating epithelial cancer cells predicted poor clinical outcome (tumor recurrence; p < 0.0001; log-rank test), and was functionally associated with FDG-PET avidity (p < 0.04). Similarly, oxidative stress (MCT4+) in tumor stromal cells was specifically associated with higher tumor stage (p < 0.03), and was a highly specific marker for cancer-associated fibroblasts (p < 0.001). We propose that oxidative stress is a key hallmark of tumor tissues that drives high-energy metabolism in adjacent proliferating mitochondrial-rich cancer cells, via the paracrine transfer of mitochondrial fuels (such as L-lactate and ketone bodies). New antioxidants and MCT4 inhibitors should be developed to metabolically target “three-compartment tumor metabolism” in head and neck cancers. It is remarkable that two “non-proliferating” populations of cells (Ki-67−/MCT4+) within the tumor can actually determine clinical outcome, likely by providing high-energy mitochondrial “fuels” for proliferative cancer cells to burn. Finally, we also show that in normal mucosal tissue, the basal epithelial “stem cell” layer is hyper-proliferative (Ki-67+), mitochondrial-rich (TOMM20+/COX+) and is metabolically programmed to use mitochondrial fuels (MCT1+), such as ketone bodies and L-lactate. Thus, oxidative mitochondrial metabolism (OXPHOS) is a common feature of both (1) normal stem cells and (2) proliferating cancer cells. As such, we should consider metabolically treating cancer patients with mitochondrial inhibitors (such as Metformin), and/or with a combination of MCT1 and MCT4 inhibitors, to target “metabolic symbiosis.”

The human ␤-globin genes are expressed in a developmental stage-specific manner in erythroid cells. Gene-proximal cis-regulatory DNA elements and interacting proteins restrict the expression of the genes to the embryonic, fetal, or adult stage of erythropoiesis. In addition, the relative order of the genes with respect to the locus control region contributes to the temporal regulation of the genes. We have previously shown that transcription factors TFII-I and USF interact with the ␤-globin promoter in erythroid cells. Herein we demonstrate that reducing the activity of USF decreased ␤-globin gene expression, while diminishing TFII-I activity increased ␤-globin gene expression in erythroid cell lines. Furthermore, a reduction of USF activity resulted in a significant decrease in acetylated H3, RNA polymerase II, and cofactor recruitment to the locus control region and to the adult ␤-globin gene. The data suggest that TFII-I and USF regulate chromatin structure accessibility and recruitment of transcription complexes in the ␤-globin gene locus and play important roles in restricting ␤-globin gene expression to the adult stage of erythropoiesis.

BackgroundWhile programmed death 1 (PD-1) and programmed death-ligand 1 (PD-L1) checkpoint inhibitors have activity in a proportion of patients with advanced bladder cancer, strongly predictive and prognostic biomarkers are still lacking. In this study, we evaluated PD-L1 protein expression on circulating tumor cells (CTCs) isolated from patients with muscle invasive (MIBC) and metastatic (mBCa) bladder cancer and explore the prognostic value of CTC PD-L1 expression on clinical outcomes.MethodsBlood samples from 25 patients with MIBC or mBCa were collected at UCSF and shipped to Epic Sciences. All nucleated cells were subjected to immunofluorescent (IF) staining and CTC identification by fluorescent scanners using algorithmic analysis. Cytokeratin expressing (CK)+ and (CK)−CTCs (CD45−, intact nuclei, morphologically distinct from WBCs) were enumerated. A subset of patient samples underwent genetic characterization by fluorescence in situ hybridization (FISH) and copy number variation (CNV) analysis.ResultsCTCs were detected in 20/25 (80 %) patients, inclusive of CK+ CTCs (13/25, 52 %), CK−CTCs (14/25, 56 %), CK+ CTC Clusters (6/25, 24 %), and apoptotic CTCs (13/25, 52 %). Seven of 25 (28 %) patients had PD-L1+ CTCs; 4 of these patients had exclusively CK−/CD45−/PD-L1+ CTCs. A subset of CTCs were secondarily confirmed as bladder cancer via FISH and CNV analysis, which revealed marked genomic instability. Although this study was not powered to evaluate survival, exploratory analyses demonstrated that patients with high PD-L1+/CD45−CTC burden and low burden of apoptotic CTCs had worse overall survival.ConclusionsCTCs are detectable in both MIBC and mBCa patients. PD-L1 expression is demonstrated in both CK+ and CK−CTCs in patients with mBCa, and genomic analysis of these cells supports their tumor origin. Here we demonstrate the ability to identify CTCs in patients with advanced bladder cancer through a minimally invasive process. This may have the potential to guide checkpoint inhibitor immune therapies that have been established to have activity, often with durable responses, in a proportion of these patients.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-016-2758-3) contains supplementary material, which is available to authorized users.

2Helix-loop-helix (HLH) proteins play a profound role in the process of development and cellular differentiation. Among the HLH proteins expressed in differentiating erythroid cells are the ubiquitous proteins Myc, USF1, USF2, and TFII-I, as well as the hematopoiesis-specific transcription factor Tal1/SCL. All of these HLH proteins exhibit distinct functions during the differentiation of erythroid cells. For example, Myc stimulates the proliferation of erythroid progenitor cells, while the USF proteins and Tal1 regulate genes that specify the differentiated phenotype. This minireview summarizes the known activities of Myc, USF, TFII-I, and Tal11/ SCL and discusses how they may function sequentially, cooperatively, or antagonistically in regulating expression programs during the differentiation of erythroid cells.Adult erythroid cells differentiate from hematopoietic stem cells (HSCs) through a cascade of steps (18,132). The most primitive HSC is called a long-term HSC (LT-HSC) for its ability to reconstitute HSCs in the bone marrow of irradiated mice over a long period of time. These slowly dividing cells are attached to a niche in the bone marrow and give rise to shortterm HSCs, which then differentiate into common lymphoid progenitors (CLPs) or common myeloid progenitors (CMPs). The CMPs go on to differentiate into granulocyte/monocyte precursors (GMPs) or into megakaryocyte/erythroid cell precursors (MEPs). MEPs further differentiate into erythropoietin-responsive BFU-E (blast-forming unit-erythroid) and then CFU-E (CFU-erythroid). The CFU-E cells differentiate to form orthochromatic normoblasts, then reticulocytes, and finally enucleated mature erythrocytes (125). The process of erythropoiesis has been extensively studied in vitro and in vivo and led to the identification of key erythroid cell transcription factors that regulate gene expression programs at the various steps of differentiation. The availability of erythroid cells representing different stages of maturation has rendered this system ideal for studying gene regulatory mechanisms.Transcription factors are classified based on the presence of specific protein-protein and protein-DNA interaction motifs which allow them to regulate gene expression by binding to DNA in a sequence-specific manner and to recruit coregulator complexes (98). The class of helix-loop-helix (HLH) transcription factors encompasses many proteins that play important roles during development and differentiation (89). The HLH motif is a characteristic dimerization domain which is accompanied by a basic (b) DNA-binding domain. Some HLH proteins contain an additional leucine zipper (ZIP) protein interaction module; these proteins are referred to as bHLHZIP proteins (89). Erythroid cells express many different HLH proteins. Here, we will review the well-characterized proteins USF1, USF2, Myc, TFII-I, and Tal1/SCL but will also discuss how inhibitor of DNA binding (ID) proteins, which only contain the HLH domain, may interfere with the function of HLH transcription factors in erythro...