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Supplementary Figure 2 from Systemic Treatment with the Antidiabetic Drug Metformin Selectively Impairs p53-Deficient Tumor Cell Growth

Supplementary Figure Legend from Chemotherapy Induces Tumor Clearance Independent of Apoptosis

The effect of the antidiabetic drug metformin on tumor growth was investigated using the paired isogenic colon cancer cell lines HCT116 p53+/+ and HCT116 p53−/−. Treatment with metformin selectively suppressed the tumor growth of HCT116 p53−/− xenografts. Following treatment with metformin, we detected increased apoptosis in p53−/− tumor sections and an enhanced susceptibility of p53−/− cells to undergo apoptosis in vitro when subject to nutrient deprivation. Metformin is proposed to function in diabetes treatment as an indirect activator of AMP-activated protein kinase (AMPK). Treatment with AICAR, another AMPK activator, also showed a selective ability to inhibit p53−/− tumor growth in vivo. In the presence of either of the two drugs, HCT116 p53+/+ cells, but not HCT116 p53−/− cells, activated autophagy. A similar p53-dependent induction of autophagy was observed when nontransformed mouse embryo fibroblasts were treated. Treatment with either metformin or AICAR also led to enhanced fatty acid β-oxidation in p53+/+ MEFs, but not in p53−/− MEFs. However, the magnitude of induction was significantly lower in metformin-treated cells, as metformin treatment also suppressed mitochondrial electron transport. Metformin-treated cells compensated for this suppression of oxidative phosphorylation by increasing their rate of glycolysis in a p53-dependent manner. Together, these data suggest that metformin treatment forces a metabolic conversion that p53−/− cells are unable to execute. Thus, metformin is selectively toxic to p53-deficient cells and provides a potential mechanism for the reduced incidence of tumors observed in patients being treated with metformin. [Cancer Res 2007;67(14):6745–52]

Supplementary Figure 1 from Chemotherapy Induces Tumor Clearance Independent of Apoptosis

Neoadjuvant chemotherapy (NACT) before radical gastrectomy is preferred for locally advanced gastric cancer (GC). However, clinical practices demonstrate that a considerable proportion of GC patients do not benefit from NACT, largely due to the lack of biomarkers for patient selection and prognosis prediction. A recent study revealed that patients with microsatellite instability-high (MSI-H) may be resistant to NACT, however, most tumors in Chinese GC patients (~ 95%) are characterized by microsatellite stability (MSS). Here, we aimed to discover new molecular biomarkers for this larger population. We performed whole-exome sequencing on 46 clinical samples (pre- and post-treatment) from 30 stage II/III MSS GC patients whose response to NACT was rigorously defined. Serum tumor markers (TMs), including AFP, CEA, CA199, CA724 and CA242 were measured during the course. High tumor mutation burden (TMB-H) and 19q12 amplification (19q12 +) were positively associated with the NACT response. When TMB and 19q12 amplification were jointly analyzed, those with TMB-H or 19q12 + showed favorable response to NACT (p = 0.035). Further, TMB-H was negatively correlated with ypN stage, lymph node metastasis, and macrophage infiltration. Patients with TMB-H showed better disease-free survival (DFS) than those with TMB-L (P = 0.025, HR = 0.1331), and this was further validated using two larger GC datasets: TCGA-STAD (p = 0.004) and ICGC-CN (p = 0.045). The combination of TMB-H and 19q12 + can serve as an early indicator of response to NACT. Superior to traditional clinical indicators, TMB-H is a robust and easily accessible candidate biomarker associated with better DFS, and can be evaluated at the time of diagnosis.

Aiming at the problems of large leakage current and low efficiency of existing transformerless single-phase grid-connected photovoltaic inverters, a novel transformerless single-phase grid-connecte...

Homozygous deletion of methylthioadenosine phosphorylase (MTAP) is one of the most frequent genetic alterations in glioblastoma (GBM), but its pathologic consequences remain unclear. In this study, we report that loss of MTAP results in profound epigenetic reprogramming characterized by hypomethylation of PROM1/CD133–associated stem cell regulatory pathways. MTAP deficiency promotes glioma stem-like cell (GSC) formation with increased expression of PROM1/CD133 and enhanced tumorigenicity of GBM cells and is associated with poor prognosis in patients with GBM. As a combined consequence of purine production deficiency in MTAP-null GBM and the critical dependence of GSCs on purines, the enriched subset of CD133+ cells in MTAP-null GBM can be effectively depleted by inhibition of de novo purine synthesis. These findings suggest that MTAP loss promotes the pathogenesis of GBM by shaping the epigenetic landscape and stemness of GBM cells while simultaneously providing a unique opportunity for GBM therapeutics. Significance: This study links the frequently mutated metabolic enzyme MTAP to dysregulated epigenetics and cancer cell stemness and establishes MTAP status as a factor for consideration in characterizing GBM and developing therapeutic strategies.

Li-Fraumeni syndrome (LFS), a rare autosomal-dominant inheritance condition, is associated with a family cancer history as well as pathogenic/likely-pathogenic TP53 germline variants (P/LP TP53 GV). The current clinical methods for detecting LFS are limited. Here, we retrospectively investigate P/LP TP53 GV among Chinese cancer patients by next-generation sequencing and evaluate its relationship with a family cancer history. A total of 270 out of 19,226 cancer patients have TP53 GV, including 53 patients with P/LP TP53 GV. Patients with P/LP TP53 GV are mainly found in male with glioma, lung cancer or sarcoma. The median age of diagnosis for P/LP TP53 GV patients is significantly lower than that of non-P/LP TP53 GV patients (31-years vs. 53-years; P 0.01). One LFS patient and 3 Li-Fraumeni-like syndrome (LFL) patients are among the 26 followed-up P/LP TP53 GV patients. Among 25 types of P/LP TP53 GV, the highest variant frequencies occurred at codon 175 and 248. p.M237I, p.R158H, p.C238Y and p.C275R, are firstly identified among the Chinese LFS/LFL patients. This study reports the (P/LP) TP53 GV characteristics of Chinese pan-cancer patients. These findings suggest analyzing the P/LP TP53 GV in cancer patients is an effective strategy for identifying cancer predisposition syndrome.

IntroductionLeptomeningeal metastasis (LM) commonly occurs in non-small cell lung cancer (NSCLC) patients and has a poor prognosis. Due to limited access to leptomeningeal lesions, the genetic characteristics of LM have not been explored to date. Cerebrospinal fluid (CSF) may be the most representative liquid biopsy medium to obtain genomic information from LM in NSCLC.MethodsCSF biopsies and matched peripheral blood biopsies were collected from 33 NSCLC patients with LM. We profiled genetic alterations from LM by comparing CSF cell-free DNA (cfDNA) with plasma cfDNA. Somatic mutations were examined using targeted sequencing. Genomic instability was analyzed by low-coverage whole-genome sequencing (WGS).ResultsDriver mutations were detected in 100% of CSF cfDNA with much higher variant allele frequency than that in matched plasma cfDNA (57.5%). Furthermore, we found that the proportions of CSF cfDNA fragments below 150 bp were significantly higher than those in plasma cfDNA. These findings indicate enrichment of circulating tumor DNA (ctDNA) in CSF and explain the high sensitivity of mutation detection in the CSF. The absence of some mutations in CSF cfDNA—especially the first-/second-generation mutation T790M, which confers resistance to epidermal growth factor receptor (EGFR)-Tyrosine kinase inhibitors (TKIs)—that were present in plasma cfDNA samples indicates different mechanisms of cancer evolution between LM and extracranial lesions. In addition, 86.6% of CSF ctDNA samples revealed high levels of genomic instability compared with 2.5% in plasma cfDNA samples. A higher number of large-scale state transitions (LSTs) in CSF cfDNA were associated with a shorter overall survival (OS).ConclusionOur results suggest that LM and extracranial lesions develop independently. Both CSF cfDNA genetic profiling and plasma cfDNA genetic profiling are necessary for clinical decision-making for NSCLC patients with LM. Through CSF-based low-coverage WGS, a high level of LSTs was identified as a potential biomarker of poor prognosis.

Purpose: This study aimed to evaluate the effect of continuous clustered care on the physical growth of preterm infants and on the satisfaction with the nursing care. Methods: 120 preterm infants who were admitted to our hospital from Jan 2018 to Dec 2018 were recruited as the study cohort. These infants were randomly divided into a control group (n=60, routine care) and an experimental group (n=60, continuous clustered nursing care). The study compared the parenting skills in the two groups, the infant development at 6 and 12 months, the prevalence of diseases within one year after discharge, and the parents’ satisfaction with the nursing care. Results: There was no significant difference in the parenting skill levels between the two groups. The body masses, heights, and head circumferences of the infants at 6-months and 12-months in the experimental group were better than they were in the control group. The experimental group’s physical and intellectual development were significantly better than the physical and intellectual development in the control group (P

Background Intracranial hemorrhage is a severe cranial disease in the perinatal period. We aimed to explore the feasibility and accuracy of three-dimensional (3D) ultrasonography for the quantitative evaluation of neonatal intracranial hemorrhage. Methods A total of 374 neonates with suspected intracranial hemorrhage from January 2017 to December 2019 were selected to be primarily screened by cranial ultrasonography and then diagnosed by cranial CT scan. The examination results were compared to analyze the feasibility and accuracy of 3D ultrasonography in quantifying blood loss. Results CT scan showed that there were 102 cases of Papile grade I, 106 cases of grade II, 124 cases of grade III and 42 cases of grade IV. 3D ultrasonography showed that there were 108 cases of Papile grade I, 98 cases of grade II, 130 cases of grade III and 38 cases of grade IV. The diagnostic results of these two methods were not significantly different (P>0.05). The accuracies of CT scan for subventricular, intraventricular, subdural, subarachnoid and intraparenchymal hemorrhages were 47.33%, 31.24%, 94.62%, 91.73% and 91.35% respectively, and those of 3D ultrasonography were 98.74%, 96.37%, 91.51%, 90.41% and 97.64% respectively. The accuracies of 3D ultrasonography were significantly superior to those of CT scan for subependymal, intraventricular and intraparenchymal hemorrhages (P Conclusions Neonatal intracranial hemorrhage can be well diagnosed by cranial ultrasonography which timely provides evidence for clinicians, thereby elevating the cure rate and reducing the mortality rate and incidence rate of sequelae. 3D ultrasonography is feasible and accurate for the quantitative evaluation of neonatal intracranial hemorrhage, thus being of great significance to prognostic determination in clinical practice.

Preterm infants constitute an important proportion of neonatal deaths and various complications, and very preterm infants (VPI) are more likely to develop severe complications, such as intraventricular hemorrhage (IVH), anemia, and sepsis. It has been confirmed that placental transfusion can supplement blood volume in infants and reduce preterm-associated complications, which is further conducive to the development of the nervous system and a better long-term prognosis. Based on these advantages, placental transfusion has been widely used in VPI. There are three main types of placental transfusion: delayed cord clamping (DCC), intact umbilical cord milking (I-UCM), and cut umbilical cord milking (C-UCM). However, the optimal method for PT-VPI remains controversial, and it is urgent to identify the best method of placental transfusion. We plan to fully evaluate the safety and effectiveness of these three placental transfusion methods in VPI in a 3-arm multicenter randomized controlled trial: Placental Transfusion in Very Preterm Infants (PT-VPI). Trial registration: chictr.org.cn, number ChiCTR2000030953.

Precise genetic counseling and prenatal diagnosis are often hindered by incomplete penetrance of risk variance and complex patterns of inheritance. Here, we performed a clinical and genetic study of a five-generation Pakistani family with a history of multiple cases of childhood brain tumors. Six affected individuals died of brain tumors at very early ages and three were confirmed as having a homozygous mutation in exon 6 of the PMS2 gene (c.543delT). Fifteen members of the family were identified as heterozygous carriers of this mutation with a lack of cancer incidence. Both clinical manifestations and genetic test results of brain tumor patients in the family support the diagnosis of constitutional mismatch repair deficiency (CMMRD) syndrome, a condition in which individuals carry homozygous germline mutations in mismatch repair machinery genes with an early onset of malignancies such as glioma. This information was used to guide prenatal diagnosis with genetic testing on chorionic villus samples for the family. This is the first report of prenatal genetic diagnosis of hereditary brain tumor.

Background Mutations in telomerase reverse transcriptase promoter (TERTp) and isocitrate dehydrogenase 1 and 2 (IDH) offer objective markers to assist in classifying diffuse gliomas into genetic subgroups. However, traditional mutation detection techniques lack sensitivity or have long turnaround times or high costs. We developed GliomaDx, an allele-specific, locked nucleic acid-based quantitative PCR assay to overcome these limitations and sensitively detect TERTp and IDH mutations. Methods We evaluated the performance of GliomaDx on cell line DNA and frozen tissue diffuse glioma samples with variable tumor percentage to mimic use in clinical settings and validated low percentage variants using sensitive techniques including droplet digital PCR (ddPCR) and next-generation sequencing. We also developed GliomaDx Nest, which incorporates a high-fidelity multiplex pre-amplification step prior to allele-specific PCR for low-input formalin-fixed paraffin embedded (FFPE) samples. Results GliomaDx detects the TERTp and IDH1 alterations at an analytical sensitivity of 0.1% mutant allele fraction, corresponding to 0.2% tumor cellularity. GliomaDx identified TERTp/IDH1 alterations in a cohort of frozen tissue samples with variable tumor percentage of all major diffuse glioma histologic types. GliomaDx Nest is able to detect these hotspot mutations with similar sensitivity from pre-amplified samples and was successfully tested on a cohort of clinical FFPE samples. Testing of a cohort of previously identified TERTpWT-IDHWT gliomas (by Sanger sequencing) revealed that 26.3% harbored low-percentage mutations. Analysis by ddPCR and whole exome sequencing of these tumors confirmed the low mutant fraction of these alterations and overall mutation-based tumor purity. Conclusions Our results show that GliomaDx can rapidly detect TERTp/IDH mutations with high sensitivity, identifying cases that might be missed due to the lack of sensitivity of other techniques. This approach may facilitate more objective classification of diffuse glioma samples in clinical settings such as intraoperative diagnosis or in testing cases with low tumor purity.

// Zhen-Yu Zhang 1, * , Aden Ka-Yin Chan 2, * , Xiao-Jie Ding 1 , Zhi-Yong Qin 1 , Christopher S. Hong 3 , Ling-Chao Chen 1 , Xin Zhang 1 , Fang-Ping Zhao 4 , Yin Wang 5 , Yang Wang 1 , Liang-Fu Zhou 1 , Zhengping Zhuang 3 , Ho-Keung Ng 2 , Hai Yan 6 , Yu Yao 1 , Ying Mao 1 1 Department of Neurosurgery, Huashan Hospital, Fudan University, Shanghai, China 2 Department of Anatomical and Cellular Pathology, Prince of Wales Hospital, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong, China 3 Surgical Neurology Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland, USA 4 Genetron Health, Inc., Chaoyang District, Beijing, China 5 Department of Neuropathology, Huashan Hospital, Fudan University, Shanghai, China 6 Department of Pathology, Duke University Medical Center, The Preston Robert Tisch Brain Tumor Center, The Pediatric Brain Tumor Foundation Institute, Durham, North Carolina, USA * These authors have contributed equally to this work Correspondence to: Yu Yao, e-mail: yu_yao@fudan.edu.cn Hai Yan, e-mail: hai.yan@duke.edu Ho-Keung Ng, e-mail: hkng@cuhk.edu.hk Keywords: TERT promoter, IDH, gliomas, radiation therapy, chemotherapy Received: March 29, 2015 Accepted: June 26, 2015 Published: July 09, 2015 ABSTRACT IDH mutations frequently occur in WHO grade II and III diffuse gliomas and have favorable prognosis compared to wild-type tumors. However, whether IDH mutations in WHO grade II and II diffuse gliomas predict enhanced sensitivity to adjuvant radiation (RT) or chemotherapy (CHT) is still being debated. Recent studies have identified recurrent mutations in the promoter region of telomerase reverse transcriptase ( TERT ) in gliomas. We previously demonstrated that TERT promoter mutations may be promising biomarkers in glioma survival prognostication when combined with IDH mutations. This study analyzed IDH and TERT promoter mutations in 295 WHO grade II and III diffuse gliomas treated with or without adjuvant therapies to explore their impact on the sensitivity of tumors to genotoxic therapies. IDH mutations were found in 216 (73.2%) patients and TERT promoter mutations were found in 112 (38%) patients. In multivariate analysis, IDH mutations ( p < 0.001) were independent prognostic factors for PFS and OS in patients receiving genotoxic therapies while TERT promoter mutations were not. In univariate analysis, IDH and TERT promoter mutations were not significant prognostic factors in patients who did not receive genotoxic therapies. Adjuvant RT and CHT were factors independently impacting PFS (RT p = 0.001, CHT p = 0.026) in IDH mutated WHO grade II and III diffuse gliomas but not in IDH wild-type group. Univariate and multivariate analyses demonstrated TERT promoter mutations further stratified IDH wild-type WHO grade II and III diffuse gliomas into two subgroups with different responses to genotoxic therapies. Adjuvant RT and CHT were significant parameters influencing PFS in the IDH wt/ TERT mut subgroup (RT p = 0.015, CHT p = 0.015) but not in the IDH wt/ TERT wt subgroup. Our data demonstrated that IDH mutated WHO grade II and III diffuse gliomas had better PFS and OS than their IDH wild-type counterparts when genotoxic therapies were administered after surgery. Importantly, we also found that TERT promoter mutations further stratify IDH wild-type WHO grade II and III diffuse gliomas into two subgroups with different responses to adjuvant therapies. Taken together, TERT promoter mutations may predict enhanced sensitivity to genotoxic therapies in IDH wild-type WHO grade II and III diffuse gliomas and may justify intensified treatment in this subgroup.

The concept that human cancer is in essence a genetic disease driven by gene mutations has been well established, yet its utilization in functional studies of cancer genes has not been fully explored. Here, we describe a simple genetics-based approach that can quickly and sensitively reveal the effect of the alteration of a gene of interest on the fate of its host cells within a heterogeneous population, essentially monitoring the genetic selection that is associated with and powers the tumorigenesis. Using this approach, we discovered that loss-of-function of TP53 can promote the development of resistance of castration in prostate cancer cells via both transiently potentiating androgen-independent cell growth and facilitating the occurrence of genome instability. The study thus reveals a novel genetic basis underlying the development of castration resistance in prostate cancer cells and provides a facile genetic approach for studying a cancer gene of interest in versatile experimental conditions.

// Cheng Xu 1, * , Xiaoqing Liu 2, 4, * , Yibo Geng 1 , Qingran Bai 2, 4 , Changcun Pan 1 , Yu Sun 1 , Xin Chen 1 , Hai Yu 1 , Yuliang Wu 1 , Peng Zhang 1 , Wenhao Wu 1 , Yu Wang 1 , Zhen Wu 1 , Junting Zhang 1 , Zhaohui Wang 3 , Rui Yang 3 , Jenna Lewis 3 , Darell Bigner 3 , Fangping Zhao 5 , Yiping He 3 , Hai Yan 3 , Qin Shen 2 and Liwei Zhang 1 1 Department of Neurosurgery, Beijing Tiantan Hospital, Capital Medical University, Beijing, China 2 Center for Life Sciences, Center for Stem Cell Biology and Regenerative Medicine, School of Medicine, Tsinghua University, Beijing, China 3 Department of Pathology, Duke University Medical Center, The Preston Robert Tisch Brain Tumor Center, The Pediatric Brain Tumor Foundation Institute, Durham, North Carolina, USA 4 Peking-Tsinghua-NIBS Graduate Program, School of Life Sciences, Tsinghua University, Beijing, China 5 Genotron Health (Beijing) Co. Ltd, Beijing, China * These authors have contributed equally to this work Correspondence to: Liwei Zhang, email: zlwttn@gmail.com Qin Shen, email: shenqin@mail.tsinghua.edu.cn Keywords: DIPG, pre-clinical model, patient-derived cell line, orthotopic xenograft, pediatric brain tumor Received: March 07, 2017 Accepted: May 22, 2017 Published: July 28, 2017 ABSTRACT Diffuse intrinsic pontine glioma (DIPG) is a devastating brain tumor, with a median survival of less than one year. Due to enormous difficulties in the acquisition of DIPG specimens and the sophisticated technique required to perform brainstem orthotopic injection, only a handful of DIPG pre-clinical models are available. In this study, we successfully established eight patient-derived DIPG cell lines, mostly derived from treatment-naive surgery or biopsy specimens. These patient-derived cell lines can be stably passaged in serum-free neural stem cell media and displayed distinct morphologies, growth rates and chromosome abnormalities. In addition, these cells retained genomic hallmarks identical to original human DIPG tumors. Notably, expression of several neural stem cell lineage markers was observed in DIPG cell lines. Moreover, three out of eight cell lines can form orthotopic tumors in mouse brainstem by stereotactic injection and these tumors faithfully represented the characteristics of human DIPG by magnetic resonance imaging (MRI) and histopathological staining. Taken together, we established DIPG pre-clinical models resembling human DIPG and they provided a valuable resource for future biological and therapeutic studies.

As chronic myeloid leukemia (CML) progresses from the chronic phase to blast crisis, the levels of BCR-ABL increase. In addition, blast-transformed leukemic cells display enhanced resistance to imatinib in the absence of BCR-ABL-resistance mutations. In this study, we show that when BCR-ABL-transformed cell lines were selected for imatinib resistance in vitro, the cells that grew out displayed a higher BCR-ABL expression comparable to the increase seen in accelerated forms of the disease. This enhanced expression of BCR-ABL was associated with an increased rate of glycolysis but with a decreased rate of proliferation. The higher level of BCR-ABL expression in the selected cells correlated with a nonhypoxic induction of hypoxia-inducible factor-1alpha (HIF-1alpha) that was required for cells to tolerate enhanced BCR-ABL signaling. HIF-1alpha induction resulted in an enhanced rate of glycolysis but with reduced glucose flux through both the tricarboxylic acid cycle and the oxidative arm of the pentose phosphate pathway (PPP). The reduction in oxidative PPP-mediated ribose synthesis was compensated by the HIF-1alpha-dependent activation of the nonoxidative PPP enzyme, transketolase, in imatinib-resistant CML cells. In both primary cultures of cells from patients exhibiting blast transformation and in vivo xenograft tumors, use of oxythiamine, which can inhibit both the pyruvate dehydrogenase complex and transketolase, resulted in enhanced imatinib sensitivity of tumor cells. Together, these results suggest that oxythiamine can enhance imatinib efficacy in patients who present an accelerated form of the disease.

Tumor cells increase the use of anabolic pathways to satisfy the metabolic requirements associated with a high growth rate. Transformed cells take up and metabolize nutrients such as glucose and glutamine at high levels that support anabolic growth. Oncogenic signaling through the PI3K/Akt and Myc pathways directly control glucose and glutamine uptake, respectively. In order to achieve elevated rates of nucleotide biosynthesis, neoplastic cells must divert carbon from PI3K/Akt-induced glycolytic flux into the non-oxidative branch of the pentose phosphate pathway to generate ribose 5-phosphate. This redirection of glucose catabolism appears to be regulated by cytoplasmic tyrosine kinases. Myc-induced glutamine metabolism also increases the abundance and activity of different rate-limiting enzymes that produce the molecular precursors required for de novo nucleotide synthesis. In this review, we will focus on recent progress in understanding of how glucose and glutamine metabolism is redirected by oncogenes in order to support de novo nucleotide biosynthesis during proliferation and how metabolic reprogramming can be potentially exploited in the development of new cancer therapies.

OBJECTIVE To report on the phenotype of an infant with central hypoventilation syndrome (CCHS) and result of PHOX2B gene mutation analysis for the purpose of genetic counseling and prenatal diagnosis. METHODS Clinical data of an infant with CCHS was collected and analyzed. Potential mutation of PHOX2B gene was analyzed by amplified fragment length polymorphism (amp-FLP) and DNA sequencing. RESULTS The patient had typical clinical features of CCHS including frequent hypoventilation during sleeping, hypoxemia and hypercapnia which could be corrected by continuous ventilatory support. She also had repeated bruising and was difficult-to-wean, but without any cardiac, pulmonary, neuromuscular or brainstem lesions. DNA sequencing and amp-FLP of the PHOX2B gene showed that the patient has carried a polyalanine expansion repeat mutation (PARM) in exon 3. A 27 bp duplication was confirmed in the repeat sequence of 20 alanines by cloned and sequenced. This has led to an expansion of the repeat tract to 29 alanines. The genotype was therefore 20/29. CONCLUSION A patient with CCHS has been described. Mutation screening of PHOX2B gene can be used as an important support for diagnosis and genetic counseling for such patients.

SummaryMany tumors display a high rate of glucose utilization, as evidenced by 18-F-2-deoxyglucose PET imaging. One potential advantage of catabolizing glucose through glycolysis at a rate that exceeds bioenergetic need is that the growing cell can redirect the excess glycolytic end product pyruvate toward lipid synthesis. Such de novo lipid synthesis is necessary for membrane production and lipid-based posttranslational modification of proteins. A key enzyme linking glucose metabolism to lipid synthesis is ATP citrate lyase (ACL), which catalyzes the conversion of citrate to cytosolic acetyl-CoA. ACL inhibition by RNAi or the chemical inhibitor SB-204990 limits in vitro proliferation and survival of tumor cells displaying aerobic glycolysis. The same treatments also reduce in vivo tumor growth and induce differentiation.

In this paper we consider whether the dependency of metazoan cells on extracellular signals to maintain cell survival results in an important barrier that must be overcome during carcinogenesis. It is now generally accepted that a major barrier to cancer comes from the inability of cells to enter and progress through the cell cycle in a cell-autonomous fashion. Most of the oncogenes studied over the last two decades contribute to the ability of the cancer cell to enter and progress through the cell cycle in the absence of the instructional signals normally imparted by extracellular growth factors. Over the last two decades, it has begun to be appreciated that there is a second potential barrier to transformation. It appears that all cells in multicellular organisms need extracellular signals not only to initiate proliferation, but also to maintain cell survival. Every cell in our body expresses the proteins necessary to execute its own death by apoptosis. A cell will activate this apoptotic program by default unless it receives signals from the extracellular environment that allow the cell to suppress the apoptotic machinery it expresses. It now appears that the molecular basis of this suppression lies in the signaling pathways that regulate cellular nutrient uptake and direct the metabolic fate of those nutrients.

By functional cloning, we have established that Ran GTPase is involved in LPS-induced signal transduction. This has been accomplished by several functional comparisons of the two cDNAs, Lpsn/Ran (or RanT/n) and Lpsd/Ran (or RanC/d), which were isolated from cDNA libraries of LPS responder and hyporesponder mice, respectively. The letter n refers to the `normal' phenotype and the letter d refers to the `deficient' phenotype. Consistent with our previous results, more animal studies indicated that adenoviral transduction of RanC/d cDNA, but not RanT/n cDNA, into sensitive mice conferred significant resistance against endotoxin challenge. Thus the incorporation of RanC/d cDNA into gene therapy protocols as a therapeutic sequence remains very attractive. At steady state, hematopoietic cells transduced with RanC/d cDNA led to about a 10-fold increase in exogenous Ran protein compared with RanT/n cDNA. Furthermore, our cumulative data suggest that a slight elevation of Ran protein in B cells enhances LPS responsiveness, but the same elevation of Ran in macrophages does not. On the other hand, a high level of overexpression of Ran in both macrophages and B cells down-regulates LPS signal transduction. Thus LPS-induced signal transduction in macrophages and B cells is likely to occur via different signaling pathways.

We recently showed that adenoviral transfer and expression of theLpsd/Ran gene isolated from endotoxin-resistant C3H/HeJ mice could protect endotoxin-sensitive mice from endotoxic shock. Elevation of proinflammatory cytokines, such as tumor necrosis factor α (TNF-α), is thought to be essential for the development of septic shock. To investigate the extent to whichLpsd/Ranaffects TNF-α production, we transduced primary macrophages from endotoxin-sensitive and -resistant mice with adenoviral vectors expressing the wild-type and the mutantLps/RancDNAs and other control genes, and compared the amount of TNF-α produced by these various transduced macrophages. Successful transfer and expression ofLpsd/RancDNA in endotoxin-sensitive C3H/HeOuJ macrophages reduced TNF-α production upon lipopolysaccharide (LPS) stimulation, as compared with macrophages transduced with vectors expressing the wild-typeLpsn/RancDNA, the green fluorescent protein gene, or thelacZgene. On the other hand, successful transfer and expression of the wild-typeLpsn/RancDNA in primary macrophages from endotoxin-resistant C3H/HeJ mice failed to induce TNF-α production to any significant extent unless a very high LPS concentration was used. Given our previous demonstration that Lpsn/Ran functions effectively in restoring LPS responsiveness in B cells from C3H/HeJ mice, we conclude thatLps/Ranis involved in a CD14-independent signal transduction pathway. This dominant negative down-regulation byLpsd/Ranon TNF-α production by macrophages and probably other innate immune responses may be key to the development of an effective gene therapy for endotoxic or septic shock.

Tumor cells are metabolically reprogrammed to fuel cell proliferation. Most transformed cells take up high levels of glucose and produce ATP through aerobic glycolysis. In cells exhibiting aerobic glycolysis, a significant fraction of glucose carbon is also directed into de novo lipogenesis and nucleotide biosynthesis. The glucose-responsive transcription factor carbohydrate responsive element binding protein (ChREBP) was previously shown to be important for redirecting glucose metabolism in support of lipogenesis in nonproliferating hepatocytes. However, whether it plays a more generalized role in reprogramming metabolism during cell proliferation has not been examined. Here, we demonstrated that the expression of ChREBP can be induced in response to mitogenic stimulation and that the induction of ChREBP is required for efficient cell proliferation. Suppression of ChREBP resulted in diminished aerobic glycolysis, de novo lipogenesis, and nucleotide biosynthesis, but stimulated mitochondrial respiration, suggesting a metabolic switch from aerobic glycolysis to oxidative phosphorylation. Cells in which ChREBP was suppressed by RNAi exhibited p53 activation and cell cycle arrest. In vivo, suppression of ChREBP led to a p53-dependent reduction in tumor growth. These results demonstrate that ChREBP plays a key role both in redirecting glucose metabolism to anabolic pathways and suppressing p53 activity.

Dysregulation of apoptosis is associated with the development of human cancer and resistance to anticancer therapy. The ultimate goal of cancer treatment is to selectively induce cancer cell death and overcome drug resistance. A deeper understanding of how a given chemotherapy affects tumor cell death is needed to develop strategically designed anticancer agents. Here, we use a xenograft mouse tumor system generated from genetically defined cells deficient in apoptosis to examine the involvement of multiple forms of cell death induced by cyclophosphamide (CP), a DNA alkylating agent commonly used in chemotherapy. We find that although apoptosis facilitates tumor regression, it is dispensable for complete tumor regression as other forms of cell death are activated. Sporadic necrosis is observed in both apoptosis-competent and deficient tumors evident by tumor cell morphology, extracellular release of high mobility group box 1 protein, and activation of innate immune cells in CP-treated tumors. Our findings indicate that in apoptosis-deficient tumors, necrosis may play a fundamental role in tumor clearance by stimulating the innate immune response. [Cancer Res 2008;68(23):9595–600]

The effect of the antidiabetic drug metformin on tumor growth was investigated using the paired isogenic colon cancer cell lines HCT116 p53+/+ and HCT116 p53−/−. Treatment with metformin selectively suppressed the tumor growth of HCT116 p53−/− xenografts. Following treatment with metformin, we detected increased apoptosis in p53−/− tumor sections and an enhanced susceptibility of p53−/− cells to undergo apoptosis in vitro when subject to nutrient deprivation. Metformin is proposed to function in diabetes treatment as an indirect activator of AMP-activated protein kinase (AMPK). Treatment with AICAR, another AMPK activator, also showed a selective ability to inhibit p53−/− tumor growth in vivo. In the presence of either of the two drugs, HCT116 p53+/+ cells, but not HCT116 p53−/− cells, activated autophagy. A similar p53-dependent induction of autophagy was observed when nontransformed mouse embryo fibroblasts were treated. Treatment with either metformin or AICAR also led to enhanced fatty acid β-oxidation in p53+/+ MEFs, but not in p53−/− MEFs. However, the magnitude of induction was significantly lower in metformin-treated cells, as metformin treatment also suppressed mitochondrial electron transport. Metformin-treated cells compensated for this suppression of oxidative phosphorylation by increasing their rate of glycolysis in a p53-dependent manner. Together, these data suggest that metformin treatment forces a metabolic conversion that p53−/− cells are unable to execute. Thus, metformin is selectively toxic to p53-deficient cells and provides a potential mechanism for the reduced incidence of tumors observed in patients being treated with metformin. [Cancer Res 2007;67(14):6745–52]

Cell proliferation requires a constant supply of lipids and lipid precursors to fuel membrane biogenesis and protein modification. Cytokine stimulation of hematopoietic cells directly stimulates glucose utilization in excess of bioenergetic demand, resulting in a shift from oxidative to glycolytic metabolism. A potential benefit of this form of metabolism is the channeling of glucose into biosynthetic pathways. Here we report that glucose supports de novo lipid synthesis in growing hematopoietic cells in a manner regulated by cytokine availability and the PI 3 K/Akt signaling pathway. The net conversion of glucose to lipid is dependent on the ability of cells to produce cytosolic acetyl CoA from mitochondria-derived citrate through the action of ATP citrate lyase (ACL). Stable knockdown of ACL leads to a significant impairment of glucose-dependent lipid synthesis and an elevation of mitochondrial membrane potential. Cells with ACL knockdown display decreased cytokine-stimulated cell proliferation. In contrast, these cells resist cell death induced by either cytokine or glucose withdrawal. However, ACL knockdown significantly impairs Akt-mediated tumorigenesis in vivo. These data suggest that enzymes involved in the conversion of glucose to lipid may be targets for the treatment of pathologic cell growth.

Purpose: A majority of human cancers have the ability to evade cell death with dysregulated cell death and survival mechanisms. This may have a direct impact on treatment resistance and tumor recurrence. This study aims to elucidate the roles of apoptosis, autophagy and necrosis induced by ionizing radiation (IR). Materials/Methods: Immortalized murine embryonic fibroblast (MEF) cell lines with various genetic backgrounds were used in the study. Xenograft tumors were generated by injecting tumor cells (engineered by retrovirally transfecting the above MEF cell lines with oncoprotein E1A and KRAS) to bilateral flanks of nude mice. Mice bearing xenograft tumors were treated with radiation by using an orthovoltage X-ray machine. Tissue cultures were irradiated by using a Gammacell 40 irradiator with a Cs137 source. Cell survival was analyzed with several commonly accepted methods. Morphology changes before and after treatments were analyzed by phase contrast microscopy as well as transmission electron microscopy (TEM). Western blotting and Immunohistochemistry (IHC) methods were carried out as described. Results: There was no significant difference in the level of tumor regression between xenografted tumors lacking Bax and Bak (Bax−/−Bak−/−) and WT tumors after radiation. While apoptotic cell death was observed in immortalized WT MEF cell line and xenograft tumors after irradiation, the intrinsic apoptosis pathway was not required Autophagy was found to be activated after radiation, and pharmacologic inhibition of autophagy by 3-methyladenine (3MA) resulted in higher survival fraction after radiation, suggesting a cytotoxic role of autophagy following radiation. However, there was no difference in radiation response when an essential autophagy gene ATG5 was stably knocked down, suggesting that autophagy activation may not be required for radiation induced cell death. We then investigated the role of poly(ADP)-ribose polymerase (PARP) in radiation response by using PARP-1−/− immortalized MEFs. These cells were more resistant to MAF (an active metabolite of cyclophosphamide) induced necrosis as was shown previously, but were more sensitive to radiation both in vitro and in vivo (as in xenograft). We also found that a specific PARP inhibitor ABT-888 can significantly sensitize xenograft tumors to radiation treatment. Conclusion: While apoptosis and autophagy occur after radiation, the intrinsic apoptosis and autophagic pathways is not required for tumor regression following IR. Conversely, PARP protein plays an important pro-survival role after IR, and deficiency of PARP renders cells more sensitive to radiation. 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 2513. doi:10.1158/1538-7445.AM2011-2513