Slavica Tudzarova

Molecular basis for the differential use of glucose and glutamine in cell proliferation as revealed by synchronized HeLa cells

During cell division, the activation of glycolysis is tightly regulated by the action of two ubiquitin ligases, anaphase-promoting complex/cyclosome–Cdh1 (APC/C-Cdh1) and SKP1/CUL-1/F-box protein–β-transducin repeat-containing protein (SCF-β-TrCP), which control the transient appearance and metabolic activity of the glycolysis-promoting enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase, isoform 3 (PFKFB3). We now demonstrate that the breakdown of PFKFB3 during S phase occurs specifically via a distinct residue (S273) within the conserved recognition site for SCF-β-TrCP. Glutaminase 1 (GLS1), the first enzyme in glutaminolysis, is also targeted for destruction by APC/C-Cdh1 and, like PFKFB3, accumulates after the activity of this ubiquitin ligase decreases in mid-to-late G1. However, our results show that GLS1 differs from PFKFB3 in that its recognition by APC/C-Cdh1 requires the presence of both a Lys-Glu-Asn box (KEN box) and a destruction box (D box) rather than a KEN box alone. Furthermore, GLS1 is not a substrate for SCF-β-TrCP and is not degraded until cells progress from S to G2/M. The presence of PFKFB3 and GLS1 coincides with increases in generation of lactate and in utilization of glutamine, respectively. The contrasting posttranslational regulation of PFKFB3 and GLS1, which we have verified by studies of ubiquitination and protein stability, suggests the different roles of glucose and glutamine at distinct stages in the cell cycle. Indeed, experiments in which synchronized cells were deprived of either of these substrates show that both glucose and glutamine are required for progression through the restriction point in mid-to-late G1, whereas glutamine is the only substrate essential for the progression through S phase into cell division.

Two ubiquitin ligases, APC/C-Cdh1 and SKP1-CUL1-F (SCF)-beta-TrCP, sequentially regulate glycolysis during the cell cycle.

During cell proliferation, the abundance of the glycolysis-promoting enzyme, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase, isoform 3 (PFKFB3), is controlled by the ubiquitin ligase APC/C-Cdh1 via a KEN box. We now demonstrate in synchronized HeLa cells that PFKFB3, which appears in mid-to-late G1, is essential for cell division because its silencing prevents progression into S phase. In cells arrested by glucose deprivation, progression into S phase after replacement of glucose occurs only when PFKFB3 is present or is substituted by the downstream glycolytic enzyme 6-phosphofructo-1-kinase. PFKFB3 ceases to be detectable during late G1/S despite the absence of Cdh1; this disappearance is prevented by proteasomal inhibition. PFKFB3 contains a DSG box and is therefore a potential substrate for SCF-β-TrCP, a ubiquitin ligase active during S phase. In synchronized HeLa cells transfected with PFKFB3 mutated in the KEN box, the DSG box, or both, we established the breakdown routes of the enzyme at different stages of the cell cycle and the point at which glycolysis is enhanced. Thus, the presence of PFKFB3 is tightly controlled to ensure the up-regulation of glycolysis at a specific point in G1. We suggest that this up-regulation of glycolysis and its associated events represent the nutrient-sensitive restriction point in mammalian cells.

Cdc7 Kinase Is a Predictor of Survival and a Novel Therapeutic Target in Epithelial Ovarian Carcinoma

Purpose: There is a lack of prognostic and predictive biomarkers in epithelial ovarian carcinoma, and the targeting of oncogenic signaling pathways has had limited impact on patient survival in this highly heterogeneous disease. The origin licensing machinery, which renders chromosomes competent for DNA replication, acts as a convergence point for upstream signaling pathways. We tested the hypothesis that Cdc7 kinase, a core component of the licensing machinery, is predictive of clinical outcome and may constitute a novel therapeutic target in epithelial ovarian carcinoma. Experimental Design: A total of 143 cases of ovarian cancer and 5 cases of normal ovary were analyzed for Cdc7 protein expression dynamics and clinicopathologic features. To assess the therapeutic potential of Cdc7, expression was down-regulated by RNA interference in SKOV-3 and Caov-3 ovarian cancer cells. Results: Increased Cdc7 protein levels were significantly associated with arrested tumor differentiation (P = 0.004), advanced clinical stage (P = 0.01), genomic instability (P < 0.001), and accelerated cell cycle progression. Multivariate analysis shows that Cdc7 predicts disease-free survival independent of patient age, tumor grade and stage (hazard ratio, 2.03; confidence interval, 1.53-2.68; P < 0.001), with the hazard ratio for relapse increasing to 10.90 (confidence interval, 4.07-29.17) for the stages 3 to 4/upper Cdc7 tertile group relative to stages 1 to 2/lower Cdc7 tertile tumors. In SKOV-3 and Caov-3 cells, Cdc7 siRNA knockdown triggered high levels of apoptosis, whereas untransformed cells arrest in G1 phase and remain viable. Conclusions: Our findings show that Cdc7 kinase predicts survival and is a potent anticancer target in epithelial ovarian carcinoma, highlighting its potential as a predictor of susceptibility to small molecule kinase inhibitors currently in development.

Molecular architecture of the DNA replication origin activation checkpoint

Perturbation of DNA replication initiation arrests human cells in G1, pointing towards an origin activation checkpoint. We used RNAi against Cdc7 kinase to inhibit replication initiation and dissect this checkpoint in fibroblasts. We show that the checkpoint response is dependent on three axes coordinated through the transcription factor FoxO3a. In arrested cells, FoxO3a activates the ARF‐∣Hdm2‐∣p53 → p21 pathway and mediates p15INK4B upregulation; p53 in turn activates expression of the Wnt/β‐catenin signalling antagonist Dkk3, leading to Myc and cyclin D1 downregulation. The resulting loss of CDK activity inactivates the Rb‐E2F pathway and overrides the G1‐S transcriptional programme. Fibroblasts concomitantly depleted of Cdc7/FoxO3a, Cdc7/p15, Cdc7/p53 or Cdc7/Dkk3 can bypass the arrest and proceed into an abortive S phase followed by apoptosis. The lack of redundancy between the checkpoint axes and reliance on several tumour suppressor proteins commonly inactivated in human tumours provides a mechanistic basis for the cancer‐cell‐specific killing observed with emerging Cdc7 inhibitors.

Subcellular proteomics reveals a role for nucleo-cytoplasmic trafficking at the DNA replication origin activation checkpoint.

Depletion of DNA replication initiation factors such as CDC7 kinase triggers the origin activation checkpoint in healthy cells and leads to a protective cell cycle arrest at the G1 phase of the mitotic cell division cycle. This protective mechanism is thought to be defective in cancer cells. To investigate how this checkpoint is activated and maintained in healthy cells, we conducted a quantitative SILAC analysis of the nuclear- and cytoplasmic-enriched compartments of CDC7-depleted fibroblasts and compared them to a total cell lysate preparation. Substantial changes in total abundance and/or subcellular location were detected for 124 proteins, including many essential proteins associated with DNA replication/cell cycle. Similar changes in protein abundance and subcellular distribution were observed for various metabolic processes, including oxidative stress, iron metabolism, protein translation and the tricarboxylic acid cycle. This is accompanied by reduced abundance of two karyopherin proteins, suggestive of reduced nuclear import. We propose that altered nucleo-cytoplasmic trafficking plays a key role in the regulation of cell cycle arrest. The results increase understanding of the mechanisms underlying maintenance of the DNA replication origin activation checkpoint and are consistent with our proposal that cell cycle arrest is an actively maintained process that appears to be distributed over various subcellular locations.

Cdc7 is a potent anti-cancer target in pancreatic cancer due to abrogation of the DNA origin activation checkpoint

Purpose Cdc7 is a serine/threonine kinase which is responsible for the ‘firing’ of replication origins leading to initiation of DNA replication. Inhibition or depletion of Cdc7 in normal cells triggers a DNA origin activation checkpoint causing a reversible G1 arrest. Here we investigate Cdc7 as a novel therapeutic target in pancreatic cancer. Experimental design Cdc7 target validation was performed by immunoexpression profiling in a cohort of 73 patients with pancreatic adenocarcinoma including 24 controls. Secondly Cdc7 kinase was targeted in Capan-1 and PANC-1 pancreatic cancer cell line models using either an siRNA against Cdc7 or alternatively a small molecule inhibitor (SMI) of Cdc7 (PHA-767491). Results Cdc7 was significantly overexpressed in pancreatic adenocarcinoma compared to benign pancreatic tissue (median LI 34.3% vs. 1.3%; P<0.0001). Cdc7 knockdown using siRNA in Capan-1 and PANC-1 cells resulted in marked apoptotic cell death when compared with control cells. A prominent sub-G1 peak was seen on flow cytometry (sub-G1 51% vs. 3% and 45% vs. 0.7% in Capan-1 and PANC-1 cells, respectively). Annexin V labelling confirmed apoptosis in 64% vs. 11% and 75% vs. 8%, respectively. Western blotting showed cleavage of PARP-1 and caspase-3 and presence of γH2A.X. TUNEL assay showed strong staining in treated cells. These results were mirrored following Cdc7 kinase inhibition with PHA-767491. Conclusions Our findings show that Cdc7 is a potent anti-cancer target in pancreatic adenocarcinoma and that Cdc7 immunoexpression levels might be used as a companion diagnostic to predict response to therapeutic siRNAs or SMIs directed against this kinase.

Quantitative Proteomics Reveals a "Poised Quiescence" Cellular State after Triggering the DNA Replication Origin Activation Checkpoint

An origin activation checkpoint has recently been discovered in the G1 phase of the mitotic cell cycle, which can be triggered by loss of DNA replication initiation factors such as the Cdc7 kinase. Insufficient levels of Cdc7 activate cell cycle arrest in normal cells, whereas cancer cells appear to lack this checkpoint response, do not arrest, and proceed with an abortive S phase, leading to cell death. The differential response between normal and tumor cells at this checkpoint has led to widespread interest in the development of pharmacological Cdc7 inhibitors as novel anticancer agents. We have used RNAi against Cdc7 in combination with SILAC-based high resolution MS proteomics to investigate the cellular mechanisms underlying the maintenance of the origin activation checkpoint in normal human diploid fibroblasts. Bioinformatics analysis identified clear changes in wide-ranging biological processes including altered cellular energetic flux, moderate stress response, reduced proliferative capacity, and a spatially distributed response across the mitochondria, lysosomes, and the cell surface. These results provide a quantitative overview of the processes involved in maintenance of the arrested state, show that this phenotype involves active rather than passive cellular adaptation, and highlight a diverse set of proteins responsible for cell cycle arrest and ultimately for promotion of cellular survival. We propose that the Cdc7-depleted proteome maintains cellular arrest by initiating a dynamic quiescence-like response and that the complexities of this phenotype will have important implications for the continued development of promising Cdc7-targeted cancer therapies.

Exploring the interaction between siRNA and the SMoC biomolecule transporters: Implications for small molecule-mediated delivery of siRNA

The small molecule carrier class of biomolecule transporters, modeled on the third helix of the Antennapedia homeodomain, has previously been shown to transport active proteins into cells. Here, we show an improved synthetic route to small molecule carriers, including Molander chemistry using trifluoroborate salts to improve the yield of the Suzuki–Miyaura coupling step for the formation of the biphenyl backbone. The required boronic acids could be formed by the reaction of a 2‐(dimethylamino)ethyl ether‐modified aryl Grignard reagent with triisopropyl borate. The potential for the use of small molecule carriers as oligonucleotide‐transporting agents was also explored by characterizing the interactions between small molecule carriers and siRNA. Molecular dynamics and NMR analysis indicated that the small molecule carrier guanidines are stabilized by π‐cation interactions with the biphenyl system, thus not only increasing the basicity or pKa but also shielding the charge. The binding affinities of various small molecule carriers for siRNA were investigated using isothermal calorimetry and gel shift assays. Small molecule carrier‐mediated siRNA delivery to cultured fibroblasts is demonstrated, showing that small molecule carriers possess the ability to transport functional siRNA into cells. Knockdown of Cdc7 kinase, a target for cancer, is achieved.

p53 controls CDC7 levels to reinforce G1 cell cycle arrest upon genotoxic stress.

ABSTRACT DNA replication initiation is a key event in the cell cycle, which is dependent on 2 kinases - CDK2 and CDC7. Here we report a novel mechanism in which p53 induces G1 checkpoint and cell cycle arrest by downregulating CDC7 kinase in response to genotoxic stress. We demonstrate that p53 controls CDC7 stability post-transcriptionally via miR-192/215 and post-translationally via Fbxw7β E3 ubiquitin ligase. The p53-dependent pathway of CDC7 downregulation is interlinked with the p53-p21-CDK2 pathway, as p21-mediated inhibition of CDK2-dependent phosphorylation of CDC7 on Thr376 is required for GSK3ß-phosphorylation and Fbxw7ß-dependent degradation of CDC7. Notably, sustained oncogenic high levels of active CDC7 exert a negative feedback onto p53, leading to unrestrained S-phase progression and accumulation of DNA damage. Thus, p53-dependent control of CDC7 levels is essential for blocking G1/S cell-cycle transition upon genotoxic stress, thereby safeguarding the genome from instability and thus representing a novel general stress response.

The double trouble of metabolic diseases: the diabetes–cancer link

The recent recognition of the clinical association between type 2 diabetes (T2D) and several types of human cancer has been further highlighted by reports of antidiabetic drugs treating or promoting cancer. At the cellular level, a plethora of molecules operating within distinct signaling pathways suggests cross-talk between the multiple pathways at the interface of the diabetes–cancer link. Additionally, a growing body of emerging evidence implicates homeostatic pathways that may become imbalanced during the pathogenesis of T2D or cancer or that become chronically deregulated by prolonged drug administration, leading to the development of cancer in diabetes and vice versa. This notion underscores the importance of combining clinical and basic mechanistic studies not only to unravel mechanisms of disease development but also to understand mechanisms of drug action. In turn, this may help the development of personalized strategies in which drug doses and administration durations are tailored to individual cases at different stages of the disease progression to achieve more efficacious treatments that undermine the diabetes–cancer association.