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Summary Duchenne muscular dystrophy (DMD) is a rare X-linked recessive disease that is associated with severe progressive muscle degeneration culminating in death due to cardiorespiratory failure. We previously observed an unexpected proliferation-independent telomere shortening in cardiomyocytes of a DMD mouse model. Here, we provide mechanistic insights using human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Using traction force microscopy, we show that DMD hiPSC-CMs exhibit deficits in force generation on fibrotic-like bioengineered hydrogels, aberrant calcium handling, and increased reactive oxygen species levels. Furthermore, we observed a progressive post-mitotic telomere shortening in DMD hiPSC-CMs coincident with downregulation of shelterin complex, telomere capping proteins, and activation of the p53 DNA damage response. This telomere shortening is blocked by blebbistatin, which inhibits contraction in DMD cardiomyocytes. Our studies underscore the role of fibrotic stiffening in the etiology of DMD cardiomyopathy. In addition, our data indicate that telomere shortening is progressive, contraction dependent, and mechanosensitive, and suggest points of therapeutic intervention., Highlights • DMD hiPSC-CMs exhibit aberrant calcium handling and defective force generation • DMD hiPSC-CMs undergo proliferation-independent telomere shortening • Telomere shortening activates the p53 DNA damage pathway • Telomere shortening in DMD hiPSC-CMs is contraction dependent, In this article, Chang, Blau, and colleagues show that Duchenne muscular dystrophy (DMD) iPSC-derived cardiomyocytes exhibit proliferation-independent telomere shortening, p53 activation and mitochondrial dysfunction.

Hypoxia-induced upregulation of HIF1α triggers adipose tissue dysfunction and insulin resistance in obese patients. HIF1α closely interacts with PPARγ, the master regulator of adipocyte differentiation and lipid accumulation, but there are conflicting results how this co-regulation controls the excessive lipid accumulation that drives adipocyte dysfunction. Using single-cell imaging and modeling, we find that, surprisingly, HIF1α both promotes and represses lipid accumulation during adipogenesis. We show that the opposing roles of HIF1α are isolated from each other and depend on when HIF1α increases relative to the positive-feedback mediated upregulation of PPARγ that drives adipocyte differentiation. A theoretical model incorporating our findings resolves conflicting prior results and suggests that three network nodes before and after the isolation step have to be synergistically targeted in therapeutic strategies to revert hypoxia-mediated adipose tissue dysfunction in obesity.TeaserA systems biology approach detangles the effect of hypoxic and adipogenic regulators on lipid accumulation in adipocytes.

Background To circumvent Warburg effect, several clinical trials for different cancers are utilising a combinatorial approach using metabolic reprogramming and chemotherapeutic agents including metformin. The majority of these metabolic interventions work via indirectly activating AMP-activated protein kinase (AMPK) to alter cellular metabolism in favour of oxidative phosphorylation over aerobic glycolysis. The effect of these drugs is dependent on glycaemic and insulin conditions. Therefore, development of small molecules, which can activate AMPK, irrespective of the energy state, may be a better approach for triple-negative breast cancer (TNBC) treatment. Methods Therapeutic effect of SU212 on TNBC cells was examined using in vitro and in vivo models. Results We developed and characterised the efficacy of novel AMPK activator (SU212) that selectively induces oxidative phosphorylation and decreases glycolysis in TNBC cells, while not affecting these pathways in normal cells. SU212 accomplished this metabolic reprogramming by activating AMPK independent of energy stress and irrespective of the glycaemic/insulin state. This leads to mitotic phase arrest and apoptosis in TNBC cells. In vivo, SU212 inhibits tumour growth, cancer progression and metastasis. Conclusions SU212 directly activates AMPK in TNBC cells, but does not hamper glucose metabolism in normal cells. Our study provides compelling preclinical data for further development of SU212 for the treatment of TNBC.

Depletion of mitochondrial copper, which shifts metabolism from respiration to glycolysis and reduces energy production, is known to be effective against cancer types that depend on oxidative phosphorylation. However, existing copper chelators are too toxic or ineffective for cancer treatment. Here we develop a safe, mitochondria-targeted, copper-depleting nanoparticle (CDN) and test it against triple-negative breast cancer (TNBC). We show that CDNs decrease oxygen consumption and oxidative phosphorylation, cause a metabolic switch to glycolysis and reduce ATP production in TNBC cells. This energy deficiency, together with compromised mitochondrial membrane potential and elevated oxidative stress, results in apoptosis. CDNs should be less toxic than existing copper chelators because they favorably deprive copper in the mitochondria in cancer cells instead of systemic depletion. Indeed, we demonstrate low toxicity of CDNs in healthy mice. In three mouse models of TNBC, CDN administration inhibits tumor growth and substantially improves survival. The efficacy and safety of CDNs suggest the potential clinical relevance of this approach.

Circulating corticosteroids orchestrate stress adaptation, including inhibition of inflammation. While pathways governing corticosteroid biosynthesis and intracellular signaling are well understood, less is known about mechanisms controlling plasma corticosteroid transport. Here, we show that hepatocyte KLF15 (Kruppel-like factor 15) controls plasma corticosteroid transport and inflammatory responses through direct transcriptional activation of Serpina6 , which encodes corticosteroid-binding globulin (CBG). Klf15 -deficient mice have profoundly low CBG, reduced plasma corticosteroid binding capacity, and heightened mortality during inflammatory stress. These defects are completely rescued by reconstituting CBG, supporting that KLF15 works primarily through CBG to control plasma corticosterone homeostasis. To understand transcriptional mechanisms, we generated the first KLF15 cistromes using newly engineered Klf15 3xFLAG mice. Unexpectedly, liver KLF15 is predominantly promoter enriched, including Serpina6 , where it binds a palindromic GC-rich motif, opens chromatin, and transactivates genes with minimal associated direct gene repression. Overall, we provide critical mechanistic insight into KLF15 function and identify a hepatocyte-intrinsic transcriptional module that potently regulates systemic corticosteroid transport and inflammation.

Hypoxia plays a critical role in tumor progression including invasion and metastasis. To determine critical genes regulated by hypoxia that promote invasion and metastasis, we screen fifty hypoxia inducible genes for their effects on invasion. In this study, we identify v-maf musculoaponeurotic fibrosarcoma oncogene homolog F (MAFF) as a potent regulator of tumor invasion without affecting cell viability. MAFF expression is elevated in metastatic breast cancer patients and is specifically correlated with hypoxic tumors. Combined ChIP- and RNA-sequencing identifies IL11 as a direct transcriptional target of the heterodimer between MAFF and BACH1, which leads to activation of STAT3 signaling. Inhibition of IL11 results in similar levels of metastatic suppression as inhibition of MAFF. This study demonstrates the oncogenic role of MAFF as an activator of the IL11/STAT3 pathways in breast cancer., Hypoxia plays a critical role in tumor progression including invasion and metastasis. Here, the authors screened several hypoxia inducible genes and identified the oncogenic role of MAFF in breast cancer metastasis and that it activates IL11/STAT3 pathway.

Despite the fact that Otto H. Warburg discovered the Warburg effect almost one hundred years ago, why cancer cells waste most of the glucose carbon as lactate remains an enigma. Warburg proposed a connection between the Warburg effect and cell dedifferentiation. Hypoxia is a common tumor microenvironmental stress that induces the Warburg effect and blocks tumor cell differentiation. The underlying mechanism by which this occurs is poorly understood, and no effective therapeutic strategy has been developed to overcome this resistance to differentiation. Using a neuroblastoma differentiation model, we discovered that hypoxia repressed cell differentiation through reducing cellular acetyl-CoA levels, leading to reduction of global histone acetylation and chromatin accessibility. The metabolic switch triggering this global histone hypoacetylation was the induction of pyruvate dehydrogenase kinases (PDK1 and PDK3). Inhibition of PDKs using dichloroacetate (DCA) restored acetyl-CoA generation and histone acetylation under hypoxia. Knocking down PDK1 induced neuroblastoma cell differentiation, highlighting the critical role of PDK1 in cell fate control. Importantly, acetate or glycerol triacetate (GTA) supplementation restored differentiation markers expression and neuron differentiation under hypoxia. Moreover, ATAC-Seq analysis demonstrated that hypoxia treatment significantly reduced chromatin accessibility at RAR/RXR binding sites, which can be restored by acetate supplementation. In addition, hypoxia-induced histone hypermethylation by increasing 2-hydroxyglutarate (2HG) and reducing α-ketoglutarate (αKG). αKG supplementation reduced histone hypermethylation upon hypoxia, but did not restore histone acetylation or differentiation markers expression. Together, these findings suggest that diverting pyruvate flux away from acetyl-CoA generation to lactate production is the key mechanism that Warburg effect drives dedifferentiation and tumorigenesis. We propose that combining differentiation therapy with acetate/GTA supplementation might represent an effective therapy against neuroblastoma.

The Warburg effect is a metabolic hallmark of all cancer cells, characterized by increased glucose uptake and glycolysis for lactate generation. The generation and excretion of lactate would appear be a waste of carbon backbone and energy that is needed for proliferation. It was proposed by Warburg that the cause and consequence of the Warburg effect were the injury of respiration and cell dedifferentiation, respectively. One common factor that damages mitochondrial respiration is hypoxia, which is a metabolic stress that blocks cell differentiation and promotes cancer progression. The underlying mechanism by which this occurs is poorly understood, and no effective therapeutic strategy has been developed to overcome this resistance to differentiation. Using a neuroblastoma (NB) differentiation model, we have discovered that hypoxia represses the differentiation induced by retinoic acid (RA) as demonstrated by loss of neuron differentiation markers and changes in cell morphology, associated with reduction of global histone acetylation, that are caused by the induction of pyruvate dehydrogenase kinases (PDKs). PDKs phosphorylate pyruvate dehydrogenase (PDH), thereby blocking pyruvate entry into the TCA cycle, reducing acetyl-CoA generation, and promoting the Warburg effect. Genetic and pharmaceutical inhibition of PDK restores histone acetylation and NB cell differentiation morphology. Acetate supplementation restores histone acetylation, along with differentiation markers expression and neuron differentiation. In addition, ATAC-Seq analysis demonstrated that hypoxia treatment significantly reduces chromatin accessibility at RAR/RXR binding sites, which can be restored by acetate supplementation. These findings suggest that (1) combining RA and acetate supplementation represents a potentially effective therapeutic strategy for neuroblastoma treatment; (2) diverting pyruvate away from acetyl-CoA generation is a key mechanism by which the Warburg effect blocks cell differentiation. Citation Format: Jiangbin Ye, Yang Li, Joshua J. Gruber, Ulrike M. Litzenburger, Yiren Zhou, Yu R. Miao, Edward L. LaGory, Albert M. Li, Zhen Hu, Lori S. Hart, John M. Maris, Howard Y. Chang, Amato J. Giaccia. Deciphering Warburg effect: hypoxia inhibits tumor cell differentiation through reducing acetyl-CoA generation and chromatin accessibility [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5708.

Hypoxia (pO2 ≤ ~1.5%) is an important characteristic of tumor microenvironments that directly correlates with resistance against first-line therapies and tumor proliferation/infiltration. The ability to accurately identify hypoxic tumor cells/tissue could afford tailored therapeutic regimens for personalized treatment, development of more-effective therapies, and discerning the mechanisms underlying disease progression. Fluorogenic constructs identifying aforesaid cells/tissue operate by targeting the bioreductive activity of primarily nitroreductases (NTRs), but collectively present photophysical and/or physicochemical shortcomings that could limit effectiveness. To overcome these limitations, we present the rational design, development, and evaluation of the first activatable ultracompact xanthene core-based molecular probe (NO 2 -Rosol) for selectively imaging NTR activity that affords an "OFF-ON" near-infrared (NIR) fluorescence response (> 700 nm) alongside a remarkable Stokes shift (> 150 nm) via NTR activity-facilitated modulation to its energetics whose resultant interplay discontinues an intramolecular d-PET fluorescence-quenching mechanism transpiring between directly-linked electronically-uncoupled π-systems comprising its components. DFT calculations guided selection of a suitable fluorogenic scaffold and nitroaromatic moiety candidate that when adjoined could (i) afford such photophysical response upon bioreduction by upregulated NTR activity in hypoxic tumor cells/tissue and (ii) employ a retention mechanism strategy that capitalizes on an inherent physical property of the NIR fluorogenic scaffold for achieving signal amplification. NO 2 -Rosol demonstrated 705 nm NIR fluorescence emission and 157 nm Stokes shift, selectivity for NTR over relevant bioanalytes, and a 28-/12-fold fluorescence enhancement in solution and between cells cultured under different oxic conditions, respectively. In establishing feasibility for NO 2 -Rosol to provide favorable contrast levels in solutio/vitro, we anticipate NO 2 -Rosol doing so in preclinical studies.

Here we introduce Protein-indexed Assay of Transposase Accessible Chromatin with sequencing (Pi-ATAC) that combines single-cell chromatin and proteomic profiling. In conjunction with DNA transposition, the levels of multiple cell surface or intracellular protein epitopes are recorded by index flow cytometry and positions in arrayed microwells, and then subject to molecular barcoding for subsequent pooled analysis. PiATAC simultaneously identifies the epigenomic and proteomic heterogeneity in individual cells. Pi-ATAC reveals a casual link between transcription factor abundance and DNA motif access, and deconvolute cell types and states in the tumor microenvironment in vivo. We identify a dominant role for hypoxia, marked by HIF1A protein, in the tumor microvenvironment for shaping the regulome in a subset of epithelial tumor cells.

Summary Oxygen (O2) is both an indispensable metabolic substrate and a regulatory signal that controls the activity of Hypoxia-Inducible Factor 1α (Hif1a), a mediator of the cellular adaptation to low O2 tension (hypoxia). Hypoxic cells require Hif1a to survive. Additionally, Hif1a is an inhibitor of mitochondrial respiration. Hence, we hypothesized that enhancing mitochondrial respiration is detrimental to the survival of hypoxic cells in vivo. We tested this hypothesis in the fetal growth plate, which is hypoxic. Our findings show that mitochondrial respiration is dispensable for survival of growth plate chondrocytes. Furthermore, its impairment prevents the extreme hypoxia and the massive chondrocyte death observed in growth plates lacking Hif1a. Consequently, augmenting mitochondrial respiration affects the survival of hypoxic chondrocytes by, at least in part, increasing intracellular hypoxia. We thus propose that partial suppression of mitochondrial respiration is crucial during development to protect the tissues that are physiologically hypoxic from lethal intracellular anoxia.

Here we introduce Protein-indexed Assay of Transposase Accessible Chromatin with sequencing (Pi-ATAC) that combines single-cell chromatin and proteomic profiling. In conjunction with DNA transposition, the levels of multiple cell surface or intracellular protein epitopes are recorded by index flow cytometry and positions in arrayed microwells, and then subject to molecular barcoding for subsequent pooled analysis. Pi-ATAC simultaneously identifies the epigenomic and proteomic heterogeneity in individual cells. Pi-ATAC reveals a casual link between transcription factor abundance and DNA motif access, and deconvolute cell types and states in the tumor microenvironment in vivo. We identify a dominant role for hypoxia, marked by HIF1α protein, in the tumor microvenvironment for shaping the regulome in a subset of epithelial tumor cells., Cellular heterogeneity in cancer is complex and difficult to study. Here, the authors introduce Protein-indexed Assay of Transposase Accessible Chromatin (Pi-ATAC), which combines single cell chromatin and proteomic profiling to provide deep insight into the tumor microenvironment, and reveal the role of hypoxia in shaping the regulome of a subset of breast cancer cells in vivo.

SummaryThe p53 tumor suppressor plays a key role in maintaining cellular integrity. In response to diverse stress signals, p53 can trigger apoptosis to eliminate damaged cells or cell-cycle arrest to enable cells to cope with stress and survive. However, the transcriptional networks underlying p53 pro-survival function are incompletely understood. Here, we show that in oncogenic-Ras-expressing cells, p53 promotes oxidative phosphorylation (OXPHOS) and cell survival upon glucose starvation. Analysis of p53 transcriptional activation domain mutants reveals that these responses depend on p53 transactivation function. Using gene expression profiling and ChIP-seq analysis, we identify several p53-inducible fatty acid metabolism-related genes. One such gene, Acad11, encoding a protein involved in fatty acid oxidation, is required for efficient OXPHOS and cell survival upon glucose starvation. This study provides new mechanistic insight into the pro-survival function of p53 and suggests that targeting this pathway may provide a strategy for therapeutic intervention based on metabolic perturbation.

Dysregulation of the von Hippel-Lindau/hypoxia-inducible transcription factor (HIF) signaling pathway promotes clear cell renal cell carcinoma (ccRCC) progression and metastasis. The protein kinase GAS6/AXL signaling pathway has recently been implicated as an essential mediator of metastasis and receptor tyrosine kinase crosstalk in cancer. Here we establish a molecular link between HIF stabilization and induction of AXL receptor expression in metastatic ccRCC. We found that HIF-1 and HIF-2 directly activate the expression of AXL by binding to the hypoxia-response element in the AXL proximal promoter. Importantly, genetic and therapeutic inactivation of AXL signaling in metastatic ccRCC cells reversed the invasive and metastatic phenotype in vivo. Furthermore, we define a pathway by which GAS6/AXL signaling uses lateral activation of the met proto-oncogene (MET) through SRC proto-oncogene nonreceptor tyrosine kinase to maximize cellular invasion. Clinically, AXL expression in primary tumors of ccRCC patients correlates with aggressive tumor behavior and patient lethality. These findings provide an alternative model for SRC and MET activation by growth arrest-specific 6 in ccRCC and identify AXL as a therapeutic target driving the aggressive phenotype in renal clear cell carcinoma.

Duchenne muscular dystrophy (DMD) is an incurable X-linked genetic disease that is caused by a mutation in the dystrophin gene and affects one in every 3,600 boys. We previously showed that long telomeres protect mice from the lethal cardiac disease seen in humans with the same genetic defect, dystrophin deficiency. By generating the mdx4cv/mTRG2 mouse model with “humanized” telomere lengths, the devastating dilated cardiomyopathy phenotype seen in patients with DMD was recapitulated. Here, we analyze the degenerative sequelae that culminate in heart failure and death in this mouse model. We report progressive telomere shortening in developing mouse cardiomyocytes after postnatal week 1, a time when the cells are no longer dividing. This proliferation-independent telomere shortening is accompanied by an induction of a DNA damage response, evident by p53 activation and increased expression of its target gene p21 in isolated cardiomyocytes. The consequent repression of Pgc1α/β leads to impaired mitochondrial biogenesis, which, in conjunction with the high demands of contraction, leads to increased oxidative stress and decreased mitochondrial membrane potential. As a result, cardiomyocyte respiration and ATP output are severely compromised. Importantly, treatment with a mitochondrial-specific antioxidant before the onset of cardiac dysfunction rescues the metabolic defects. These findings provide evidence for a link between short telomere length and metabolic compromise in the etiology of dilated cardiomyopathy in DMD and identify a window of opportunity for preventive interventions.

A recent study describes a role for hypoxic signaling in the small intestine in the etiology of nonalcoholic fatty liver disease (NAFLD) and suggests that HIF-2α inhibitors may be an effective option for the treatment of this disease.

Bone development and homeostasis are achieved through an intricate balance between osteoblastic bone formation and osteoclastic bone resorption (Zaidi 2007; Khosla et al. 2008). Mammalian bone develops through endochondral ossification, a process that requires the coordination of osteoblasts and blood vessel invasion into the cartilaginous template to trigger the formation of the primary ossification center, highlighting the importance of angiogenesis during bone formation (Kronenberg 2003; Provot and Schipani 2005; Maes 2013). Maintenance of bone homeostasis is achieved by coupling bone formation with osteoblastic bone resorption. In particular, the RANK/RANKL/osteoprotegerin (OPG) axis contributes to this process by facilitating osteoblast-mediated osteoclastogenesis (Boyle et al. 2003). Deregulation of bone homeostasis is a hallmark of pathophysiological bone disease and occurs due to imbalances in osteogenic–angiogenic coupling or between bone-forming osteoblasts and bone resorption by osteoclasts. Increased bone results in osteopetrosis, whereas osteoporosis is associated with bone loss (Teitelbaum and Ross 2003; Seeman and Delmas 2006). Elucidating the molecular mechanisms that control bone homeostasis within the bone microenvironment is needed in order to identify novel therapeutic strategies to generate functional bone to treat chronic metabolic bone disorders where the options for treatment are still limited. The bone microenvironment is a particularly hypoxic microenvironment. Most adult tissues are well vascularized, allowing for the promotion of oxygen transport with normal median interstitial oxygen tension (pO2) values ranging from 3% to 9% (Vaupel et al. 1989). In contrast, pO2 levels in the bone range from 5%, the PHD enzymes regulate HIF protein stability by catalyzing the hydroxylation of HIF transcription factors on conserved proline residues, targeting them for proteosomal degradation by the von Hippel-Lindau (pVHL) ubiquitin ligase complex. When oxygen tensions fall below 5%, PHD enzyme activity is diminished, and HIF-1α and HIF-2α are stabilized and activate the expression of genes involved in diverse physiological conditions ranging from erythropoiesis to metabolism to angiogenesis (Semenza 2012). Thus, PHD enzymes provide a direct link between oxygen availability and the regulation of HIF transcriptional activity. While the PHD isoforms have nearly ubiquitous tissue expression, the tissue-specific role of each isoform in regulating HIF protein levels remains poorly defined. Here, we sought to dissect the physiologic role of oxygen-sensing PHD1, PHD2, and PHD3 enzymes in the regulation of bone homeostasis. For this purpose, we used a genetic approach to specifically inactivate Phd1, Phd2, and Phd3 in cells of the osteoblastic lineage using Cre-loxP-mediated recombination. In addition to coordinating osteoblastic–angiogenic coupling, we identified a previously unknown role for the PHD/HIF signaling pathway in the osteoblastic regulation of osteoclastogenesis through the direct regulation of OPG. Importantly, inactivation of selective PHD isoforms within the cells of the osteoblastic lineage inhibited bone loss in ovariectomized mice. Thus, molecular oxygen, through the PHD/HIF signaling pathway, plays a central role in bone homeostasis by controlling both angiogenesis and osteoclastogenesis, and therapeutic targeting of PHD enzymes can potentially be used for the treatment of bone disorders.

Signaling initiated by hypoxia and insulin powerfully alters cellular metabolism. The protein stability of hypoxia-inducible factor-1 alpha (Hif-1α) and Hif-2α is regulated by three prolyl hydroxylase domain–containing protein isoforms (Phd1, Phd2 and Phd3). Insulin receptor substrate-2 (Irs2) is a critical mediator of the anabolic effects of insulin, and its decreased expression contributes to the pathophysiology of insulin resistance and diabetes1. Although Hif regulates many metabolic pathways2, it is unknown whether the Phd proteins regulate glucose and lipid metabolism in the liver. Here, we show that acute deletion of hepatic Phd3, also known as Egln3, improves insulin sensitivity and ameliorates diabetes by specifically stabilizing Hif-2α, which then increases Irs2 transcription and insulin-stimulated Akt activation. Hif-2α and Irs2 are both necessary for the improved insulin sensitivity, as knockdown of either molecule abrogates the beneficial effects of Phd3 knockout on glucose tolerance and insulin-stimulated Akt phosphorylation. Augmenting levels of Hif-2α through various combinations of Phd gene knockouts did not further improve hepatic metabolism and only added toxicity. Thus, isoform-specific inhibition of Phd3 could be exploited to treat type 2 diabetes without the toxicity that could occur with chronic inhibition of multiple Phd isoforms.

A common genetic mutation found in clear cell renal cell carcinoma (CC-RCC) is the loss of the von Hippel-Lindau ( VHL ) gene, which results in stabilization of hypoxia-inducible factors (HIFs), and contributes to cancer progression and metastasis. CUB-domain-containing protein 1 (CDCP1) was shown to promote metastasis in scirrhous and lung adenocarcinomas as well as in prostate cancer. In this study, we established a molecular mechanism linking VHL loss to induction of the CDCP1 gene through the HIF-1/2 pathway in renal cancer. Also, we report that Fyn, which forms a complex with CDCP1 and mediates its signaling to PKCδ, is a HIF-1 target gene. Mechanistically, we found that CDCP1 specifically regulates phosphorylation of PKCδ, but not of focal adhesion kinase or Crk-associated substrate. Signal transduction from CDCP1 to PKCδ leads to its activation, increasing migration of CC-RCC. Furthermore, patient survival can be stratified by CDCP1 expression at the cell surface of the tumor. Taken together, our data indicates that CDCP1 protein might serve as a therapeutic target for CC-RCC.

Protein kinase Cdelta (PKCdelta) is an essential component of the intrinsic apoptotic program. Following DNA damage, such as exposure to UV radiation, PKCdelta is cleaved in a caspase-dependent manner, generating a constitutively active catalytic fragment (PKCdelta-cat), which is necessary and sufficient for keratinocyte apoptosis. We found that in addition to inducing apoptosis, expression of PKCdelta-cat caused a pronounced G(2)/M cell cycle arrest in both primary human keratinocytes and immortalized HaCaT cells. Consistent with a G(2)/M arrest, PKCdelta-cat induced phosphorylation of Cdk1 (Tyr(15)), a critical event in the G(2)/M checkpoint. Treatment with the ATM/ATR inhibitor caffeine was unable to prevent PKCdelta-cat-induced G(2)/M arrest, suggesting that PKCdelta-cat is functioning downstream of ATM/ATR in the G(2)/M checkpoint. To better understand the role of PKCdelta and PKCdelta-cat in the cell cycle response to DNA damage, we exposed wild-type and PKCdelta null mouse embryonic fibroblasts (MEFs) to UV radiation. Wild-type MEFs underwent a pronounced G(2)/M arrest, Cdk1 phosphorylation, and induction of apoptosis following UV exposure, whereas PKCdelta null MEFs were resistant to these effects. Expression of PKCdelta-green fluorescent protein, but not caspase-resistant or kinase-inactive PKCdelta, was able to restore G(2)/M checkpoint integrity in PKCdelta null MEFs. The function of PKCdelta in the DNA damage-induced G(2)/M cell cycle checkpoint may be a critical component of its tumor suppressor function.

The p53 tumor suppressor gene plays a critical role in maintaining tissue homeostasis through both transcriptionally dependent and independent mechanisms. The response of p53 is dictated by the type of stress (genotoxic, oncogenic, hypoxia, metabolic, etc.) through a complex and incompletely understood number of post-translational modifications. Wild type p53 promotes apoptosis, cell cycle arrest and senescence in response to growth-restrictive conditions, including glucose deprivation.1,2 Under such conditions, nuclear p53 has also been reported to stimulate autophagy through the transcriptional activation of genes that comprise the autophagic network, such as ULK1 and DRAM1, and modulators of autophagy, such as Sestrin2.3-6 Unlike nuclear p53, cytoplasmic p53 appears to inhibit autophagy through mechanisms that are not well understood, but appear to involve localization of p53 to the endoplasmic reticulum (ER) and binding to FIP200, the human ortholog of yeast Atg17.7 In fact, some cancer-associated forms of mutant p53 found predominantly in the cytoplasm can also inhibit autophagy.8 This negative regulation of autophagy by p53 has been proposed to act as rheostat to prevent an excessive amount of autophagy from occurring. Thus, the role of p53 in autophagy is complex and may be dependent on the autophagic stimulus and the mutational status of p53 in the cell. Studies describing the role p53 in the response to metabolic stress have focused primarily on the wild-type version of the protein. However, the vast majority of human tumors express high levels of mutant p53 protein that have acquired novel tumor-promoting functions distinct from those of wild type p53.9 To date, little is known about how mutant versions of p53 respond to growth-restrictive conditions, including the absence of glucose. In an elegant paper by Rodriguez et al.,10 the authors explored the effects of glucose restriction on the stability of a number of common cancer-associated p53 mutants and found that glucose deprivation resulted in degradation of mutant p53 protein levels. Interestingly, the negative regulation of p53 by glucose restriction was specific to mutant p53, since glucose restriction had a stabilizing effect on wild type p53. Surprisingly, the decreased levels of mutant p53 proteins were associated with rapid deacetylation and degradation through an autophagy-dependent but proteasome-independent process. Through the use of a constitutive acetylation-mimetic mutant p53, the authors demonstrated that autophagic degradation of p53 was dependent on the acetylation status of the protein. A major consequence of mutant p53 degradation in tumor cells after glucose deprivation is the loss of a critical check on the autophagic process that results in increased autophagy and leads to cell death (Fig. 1). Importantly, wild type p53 has been previously demonstrated to protect cells from glucose deprivation through induction of a reversible G1/S phase cell cycle arrest, suggesting that normal tissues will respond to glucose shortage differently than tumors harboring mutant p53.1 Figure 1. Glucose deprivation leads to autophagic degradation of mutant p53. (A) Wild type p53 is degraded by the proteasome under basal conditions but can be activated in an AMPK-dependent manner after glucose deprivation. Activated wild type ... The authors also provided evidence that mutant p53A135V knock-in mice fed a low-carbohydrate diet expressed reduced levels of the mutated transgene compared with mice fed on a normal or high-carbohydrate diet. Importantly the low-carbohydrate diet had no effect on wild-type p53 levels in multiple tissues studied. Critically, the authors also demonstrated that a low-glucose diet inhibited the tumor-forming ability of cells that possess mutant forms of p53, and that this was dependent on acetylation status of the mutant p53 protein. Taken together, these findings strongly indicate that some tumor-promoting forms of mutant p53 can be targeted for autophagic degradation through glucose restriction. These exciting results could be tested in the clinic by randomizing patients with tumors that harbor similar p53 mutations to a glucose-restrictive, low-carbohydrate diet compared with a normal diet. The expectation of such studies would be that the tumors of patients on a glucose-restrictive diet would see their tumors regress or grow more slowly than those on an unrestrictive glucose diet. However, the next step in this saga will be to see what combinations of chemotherapy or targeted therapy will be more effective against mutant p53 tumors that are glucose-restrictive.

Kidney cancer is the sixth leading cause of cancer death in the USA, and the primary tumor is frequently accompanied by metastasis to lungs, liver, brain and bones. Importantly a large number of kidney cancer cases are characterized by loss of von Hippel-Lindau (VHL) gene, which leads to stabilization of hypoxia-inducible factors (HIFs), which contribute to tumor progression and metastasis by multiple mechanisms. CUB-domain-containing protein 1 (CDCP1) was shown to be expressed on the cell surface of metastatic cell lines and to increase the number of nodules formed by lung adenocarcinoma cells and melanoma in lungs in tail vein injection experiments, enhance peritoneal dissemination of scirrhous adenocarcinoma, and to induce metastasis in the chicken embryo metastatic model. In the present study we investigated the role of CDCP1 protein in clear cell renal cell carcinoma (CC-RCC) and found it to be upregulated in this disease entity by a mechanism of VHL loss through HIFs. Interestingly, we found CDCP1 protein expressed not only on the membrane of kidney cancer cell lines, but also to be secreted into the media as a full length isoform. Importantly, in the knockdown experiments we found CDCP1 to promote CC-RCC cell migration in vitro, the process known to be the key during metastasis. Accordingly CDCP1 participates in the signal transduction pathway leading to PKCδ phosphorylation in CC-RCC, stimulating migration. The role of PKCδ in CDCP1-dependent migration was verified by the PKCδ knockdown experiments, which phenocopy the effect of CDCP1 knockdown; as well as migration rescue experiments, where the impairment of migration caused by CDCP1 downregulation was rescued by the overexpression of constitutively active mutant of PKCδ. Furthermore, we showed a correlation of CDCP1 cell surface expression in primary tumor with poor patient outcome. Finally, we found that suramin, a drug, which showed the clinical benefit for CC-RCC patients at premetastatic stages, causes the downregulation of CDCP1 at the protein level. Thus, the further investigation of the role of CDCP1 protein in kidney cancer metastasis is important to validate CDCP1 as a therapeutic target. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-357. doi:10.1158/1538-7445.AM2011-LB-357