Gene Summary

Gene:BNIP3L; BCL2 interacting protein 3 like
Aliases: NIX, BNIP3a
Summary:This gene encodes a protein that belongs to the pro-apoptotic subfamily within the Bcl-2 family of proteins. The encoded protein binds to Bcl-2 and possesses the BH3 domain. The protein directly targets mitochondria and causes apoptotic changes, including loss of membrane potential and the release of cytochrome c. [provided by RefSeq, Feb 2015]
Databases:OMIM, HGNC, Ensembl, GeneCard, Gene
Protein:BCL2/adenovirus E1B 19 kDa protein-interacting protein 3-like
Source:NCBIAccessed: 31 August, 2019


What does this gene/protein do?
Show (19)

Cancer Overview

Research Indicators

Publications Per Year (1994-2019)
Graph generated 31 August 2019 using data from PubMed using criteria.

Literature Analysis

Mouse over the terms for more detail; many indicate links which you can click for dedicated pages about the topic.

  • Antineoplastic Agents
  • Tumor Suppressor Proteins
  • Vesicular Transport Proteins
  • Cell Hypoxia
  • Microtubule-Associated Proteins
  • Cancer DNA
  • Up-Regulation
  • Cell Proliferation
  • Gene Expression Profiling
  • bcl-X Protein
  • Polymerase Chain Reaction
  • Nuclear Proteins
  • DNA Methylation
  • Transfection
  • Oligonucleotide Array Sequence Analysis
  • Membrane Proteins
  • Breast Cancer
  • Autophagy
  • Cancer Gene Expression Regulation
  • Apoptosis Regulatory Proteins
  • p53 Protein
  • Apoptosis
  • Beclin-1
  • Disease Progression
  • HIF1A
  • Vorinostat
  • Single Nucleotide Polymorphism
  • Biomarkers, Tumor
  • Kidney Cancer
  • Drug Resistance
  • rab GTP-Binding Proteins
  • Messenger RNA
  • Mitochondria
  • Pancreatic Cancer
  • Down-Regulation
  • siRNA
  • Proto-Oncogene Proteins
  • Liver Cancer
  • Cancer RNA
  • Mitochondrial Degradation
  • Chromosome 8
  • Stroke
  • Immunohistochemistry
Tag cloud generated 31 August, 2019 using data from PubMed, MeSH and CancerIndex

Specific Cancers (4)

Data table showing topics related to specific cancers and associated disorders. Scope includes mutations and abnormal protein expression.

Note: list is not exhaustive. Number of papers are based on searches of PubMed (click on topic title for arbitrary criteria used).

Latest Publications: BNIP3L (cancer-related)

Alvarez MC, Maso V, Torello CO, et al.
The polyphenol quercetin induces cell death in leukemia by targeting epigenetic regulators of pro-apoptotic genes.
Clin Epigenetics. 2018; 10(1):139 [PubMed] Free Access to Full Article Related Publications
BACKGROUND: In the present study, we investigated the molecular mechanisms underlying the pro-apoptotic effects of quercetin (Qu) by evaluating the effect of Qu treatment on DNA methylation and posttranslational histone modifications of genes related to the apoptosis pathway. This study was performed in vivo in two human xenograft acute myeloid leukemia (AML) models and in vitro using HL60 and U937 cell lines.
RESULTS: Qu treatment almost eliminates DNMT1 and DNMT3a expression, and this regulation was in part STAT-3 dependent. The treatment also downregulated class I HDACs. Furthermore, treatment of the cell lines with the proteasome inhibitor, MG132, together with Qu prevented degradation of class I HDACs compared to cells treated with Qu alone, indicating increased proteasome degradation of class I HDACS by Qu. Qu induced demethylation of the pro-apoptotic BCL2L11, DAPK1 genes, in a dose- and time-dependent manner. Moreover, Qu (50 μmol/L) treatment of cell lines for 48 h caused accumulation of acetylated histone 3 and histone 4, resulting in three- to ten fold increases in the promoter region of DAPK1, BCL2L11, BAX, APAF1, BNIP3, and BNIP3L. In addition, Qu treatment significantly increased the mRNA levels of all these genes, when compared to cells treated with vehicle only (control cells) (*p < 0.05).
CONCLUSIONS: In summary, our results showed that enhanced apoptosis, induced by Qu, might be caused in part by its DNA demethylating activity, by HDAC inhibition, and by the enrichment of H3ac and H4ac in the promoter regions of genes involved in the apoptosis pathway, leading to their transcription activation.

Hellwig S, Nix DA, Gligorich KM, et al.
Automated size selection for short cell-free DNA fragments enriches for circulating tumor DNA and improves error correction during next generation sequencing.
PLoS One. 2018; 13(7):e0197333 [PubMed] Free Access to Full Article Related Publications
Circulating tumor-derived cell-free DNA (ctDNA) enables non-invasive diagnosis, monitoring, and treatment susceptibility testing in human cancers. However, accurate detection of variant alleles, particularly during untargeted searches, remains a principal obstacle to widespread application of cell-free DNA in clinical oncology. In this study, isolation of short cell-free DNA fragments is shown to enrich for tumor variants and improve correction of PCR- and sequencing-associated errors. Subfractions of the mononucleosome of circulating cell-free DNA (ccfDNA) were isolated from patients with melanoma, pancreatic ductal adenocarcinoma, and colorectal adenocarcinoma using a high-throughput-capable automated gel-extraction platform. Using a 128-gene (128 kb) custom next-generation sequencing panel, variant alleles were on average 2-fold enriched in the short fraction (median insert size: ~142 bp) compared to the original ccfDNA sample, while 0.7-fold reduced in the fraction corresponding to the principal peak of the mononucleosome (median insert size: ~167 bp). Size-selected short fractions compared to the original ccfDNA yielded significantly larger family sizes (i.e., PCR duplicates) during in silico consensus sequence interpretation via unique molecular identifiers. Increments in family size were associated with a progressive reduction of PCR and sequencing errors. Although consensus read depth also decreased at larger family sizes, the variant allele frequency in the short ccfDNA fraction remained consistent, while variant detection in the original ccfDNA was commonly lost at family sizes necessary to minimize errors. These collective findings support the automated extraction of short ccfDNA fragments to enrich for ctDNA while concomitantly reducing false positives through in silico error correction.

Sindhu R, Manonmani HK
l-asparaginase induces intrinsic mitochondrial-mediated apoptosis in human gastric adenocarcinoma cells and impedes tumor progression.
Biochem Biophys Res Commun. 2018; 503(4):2393-2399 [PubMed] Related Publications
l-asparagine essentially regulates growth and proliferation of cancer cells. l-asparaginase is an anti-cancer enzyme that deprives the cancer cells of l-asparagine. The purpose of this study was to explore the mechanism of a novel l-asparaginase from Pseudomonas fluorescens on l-asparagine deprivation mediated anti-proliferation, apoptosis in human gastric adenocarcinoma cells and to evaluate inhibition of angiogenesis. We observed that, the presence of extracellular l-asparagine was essential for the growth of AGS cells. l-asparagine deprivation by l-asparaginase induced metabolic stress, cytotoxicity and apoptosis by G0 phase cell-cycle arrest, modulated the mitochondrial membrane integrity, accelerated caspase-3 activation and instigated DNA damage. The RT-PCR analysis of pro-apoptosis genes: bak1, bax, bbc3, bik, pmaip1, bnip3l, apaf1, casp3, casp7 and casp9 were significantly higher (P < 0.05), while anti-apoptotic markers xiap, bid, mcl1, and death receptor genes tnf and tradd were significantly down-regulated (P < 0.05). Additionally, higher protein expressions of p53, caspase-3 and TEM analysis showing modulations in mitochondria confirmed intrinsic apoptosis pathway. The enzyme impeded tumor progression through inhibition of cell migration and vascular remodelling of endothelial cells. Our findings suggests that the action of l-asparaginase alters mitochondrial membrane permeability and auxiliary activates intrinsic apoptosis. Therefore, this mechanistic approach might be considered as a targeted enzymotherapy against gastric adenocarcinoma.

Zhao JR, Cheng WW, Wang YX, et al.
Identification of microRNA signature in the progression of gestational trophoblastic disease.
Cell Death Dis. 2018; 9(2):94 [PubMed] Free Access to Full Article Related Publications
Gestational trophoblastic disease (GTD) encompasses a range of trophoblast-derived disorders. The most common type of GTD is hydatidiform mole (HM). Some of HMs can further develop into malignant gestational trophoblastic neoplasia (GTN). Aberrant expression of microRNA (miRNA) is widely reported to be involved in the initiation and progression of cancers. MiRNA expression profile also has been proved to be the useful signature for diagnosis, staging, prognosis, and response to chemotherapy. Till now, the profile of miRNA in the progression of GTD has not been determined. In this study, a total of 34 GTN and 60 complete HMs (CHM) trophoblastic tissues were collected. By miRNA array screening and qRT-PCR validating, six miRNAs, including miR-370-3p, -371a-5p, -518a-3p, -519d-3p, -520a-3p, and -934, were identified to be differentially expressed in GTN vs. CHM. Functional analyses further proved that miR-371a-5p and miR-518a-3p promoted proliferation, migration, and invasion of choriocarcinoma cells. Moreover, we demonstrated that miR-371a-5p was negatively related to protein levels of its predictive target genes BCCIP, SOX2, and BNIP3L, while miR-518a-3p was negatively related to MST1 and EFNA4. For the first time, we proved that miR-371a-5p and miR-518a-3p directly targeted to 3'-UTR regions of BCCIP and MST1, respectively. Additionally, we found that miR-371a-5p and miR-518a-3p regulated diverse pathways related to tumorigenesis and metastasis in choriocarcinoma cells. The results presented here may offer new clues to the progression of GTD and may provide diagnostic biomarkers for GTN.

Liu Y, Jiang Y, Wang N, et al.
Invalidation of mitophagy by FBP1-mediated repression promotes apoptosis in breast cancer.
Tumour Biol. 2017; 39(6):1010428317708779 [PubMed] Related Publications
Fructose-1,6-bisphosphatase 1, a rate-limiting enzyme in gluconeogenesis, was recently shown to be a tumor suppressor. However, the functions of fructose-1,6-bisphosphatase 1 in the regulation of mitophagy and apoptosis remain unknown. Here, we investigated the effects of fructose-1,6-bisphosphatase 1 on mitophagy and apoptosis as well as their underlying mechanisms in breast cancer cells. In this work, the messenger RNA and protein expression of various molecules were determined by quantitative realtime polymerase chain reaction and western blot, respectively. Gene-expression correlations were obtained from The Cancer Genome Atlas Breast Cancer database and analyzed using cBioPortal. The levels of cellular reactive oxygen species and apoptotic index were detected by flow cytometry. The mitochondrial membrane potentials were assessed with a JC-1 fluorescent sensor. Subcellular structures were observed under a transmission electron microscope. The intracellular distribution of translocase of outer membrane 20 was detected by immunofluorescence staining. Protein-protein interactions were analyzed by immunoprecipitation. Our results indicated that fructose-1,6-bisphosphatase 1 expression was negatively correlated with autophagy level in breast cancer. Fructose-1,6-bisphosphatase 1 restrained autophagy activity by increasing the level of p62 and decreasing the levels of LC3 and Beclin 1. Additionally, fructose-1,6-bisphosphatase 1 promoted cell apoptosis by upregulating the levels of intracellular ROS and expression of pro-apoptotic proteins such as cleaved PARP, cleaved Caspase 3, and Bax and downregulating the levels of anti-apoptotic proteins such as PARP, Caspase 3, and Bcl-2. Finally, fructose-1,6-bisphosphatase 1 limited the efficient removal of diseased mitochondria and reduced the messenger RNA and protein expressions of HIF-1α, BNIP3L/NIX, and BNIP3. More importantly, fructose-1,6-bisphosphatase 1 facilitated co-action between Bcl-2 and Beclin 1, which may be important in the mechanism of fructose-1,6-bisphosphatase 1-mediated mitophagy inhibition. In summary, loss of mitophagy by fructose-1,6-bisphosphatase 1-mediated repression promotes apoptosis in breast cancer.

Rosenblum F, Koenig RG, Mikhail FM, et al.
An adolescent with large cell calcifying sertoli cell tumor of the testis and undiagnosed Carney Complex: A case report.
Diagn Cytopathol. 2017; 45(7):634-639 [PubMed] Related Publications
Carney Complex (CNC) is a rare autosomal dominant condition with characteristic clinical presentation, tumor development, and unique genetic mutation. We present a unique case and literature review of CNC in which two neoplasms characteristic of this complex were initially diagnosed through cytological fine needle aspirate specimens, leading to the identification of CNC, with subsequent surgical and cytogenetic confirmation. Diagn. Cytopathol. 2017;45:634-639. © 2017 Wiley Periodicals, Inc.

Chen JL, David J, Cook-Spaeth D, et al.
Autophagy Induction Results in Enhanced Anoikis Resistance in Models of Peritoneal Disease.
Mol Cancer Res. 2017; 15(1):26-34 [PubMed] Free Access to Full Article Related Publications
Peritoneal carcinomatosis and peritoneal sarcomatosis is a potential complication of nearly all solid tumors and results in profoundly increased morbidity and mortality. Despite the ubiquity of peritoneal carcinomatosis/peritoneal sarcomatosis, there are no clinically relevant targeted therapies for either its treatment or prevention. To identify potential therapies, we developed in vitro models of peritoneal carcinomatosis/peritoneal sarcomatosis using tumor cell lines and patient-derived spheroids (PDS) that recapitulate anoikis resistance and spheroid proliferation across multiple cancer types. Epithelial- and mesenchymal-derived cancer cell lines (YOU, PANC1, HEYA8, CHLA10, and TC71) were used to generate spheroids and establish growth characteristics. Differential gene expression analyses of these spheroids to matched adherent cells revealed a consensus spheroid signature. This spheroid signature discriminates primary tumor specimens from tumor cells found in ascites of ovarian cancer patients and in our PDS models. Key in this gene expression signature is BNIP3 and BNIP3L, known regulators of autophagy and apoptosis. Elevated BNIP3 mRNA expression is associated with poor survival in ovarian cancer patients and elevated BNIP3 protein, as measured by IHC, and is also associated with higher grade tumors and shorter survival. Pharmacologic induction of autophagy with rapamycin significantly increased spheroid formation and survival while decreasing the induction of apoptosis. In contrast, the autophagy inhibitor hydroxychloroquine abrogated spheroid formation with a clear increase in apoptosis. Modulation of BNIP3 and the critical autophagy gene Beclin-1 (BECN1) also caused a significant decrease in spheroid formation. Combined, these data demonstrate how modulation of BNIP3-related autophagy, in PDS and in vitro spheroid models, alters the survival and morphology of spheroids.
IMPLICATIONS: Development of BNIP3/BNIP3L-targeting agents or autophagy-targeting agents may reduce morbidity and mortality associated with peritoneal carcinomatosis and sarcomatosis. Mol Cancer Res; 15(1); 26-34. ©2016 AACR.

Gothwal M, Wehrle J, Aumann K, et al.
A novel role for nuclear factor-erythroid 2 in erythroid maturation by modulation of mitochondrial autophagy.
Haematologica. 2016; 101(9):1054-64 [PubMed] Free Access to Full Article Related Publications
We have recently demonstrated that the transcription factor nuclear factor-erythroid 2, which is critical for erythroid maturation and globin gene expression, plays an important role in the pathophysiology of myeloproliferative neoplasms. Myeloproliferative neoplasm patients display elevated levels of nuclear factor-erythroid 2 and transgenic mice overexpressing the transcription factor develop myeloproliferative neoplasm, albeit, surprisingly without erythrocytosis. Nuclear factor-erythroid 2 transgenic mice show both a reticulocytosis and a concomitant increase in iron deposits in the spleen, suggesting both enhanced erythrocyte production and increased red blood cell destruction. We therefore hypothesized that elevated nuclear factor-erythroid 2 levels may lead to increased erythrocyte destruction by interfering with organelle clearance during erythroid maturation. We have previously shown that nuclear factor-erythroid 2 overexpression delays erythroid maturation of human hematopoietic stem cells. Here we report that increased nuclear factor-erythroid 2 levels also impede murine maturation by retarding mitochondrial depolarization and delaying mitochondrial elimination. In addition, ribosome autophagy is delayed in transgenics. We demonstrate that the autophagy genes NIX and ULK1 are direct novel nuclear factor-erythroid 2 target genes, as these loci are bound by nuclear factor-erythroid 2 in chromatin immunoprecipitation assays. Moreover, Nix and Ulk1 expression is increased in transgenic mice and in granulocytes from polycythemia vera patients. This is the first report implying a role for nuclear factor-erythroid 2 in erythroid maturation by affecting autophagy.

Pedanou VE, Gobeil S, Tabariès S, et al.
The histone H3K9 demethylase KDM3A promotes anoikis by transcriptionally activating pro-apoptotic genes BNIP3 and BNIP3L.
Elife. 2016; 5 [PubMed] Free Access to Full Article Related Publications
Epithelial cells that lose attachment to the extracellular matrix undergo a specialized form of apoptosis called anoikis. Here, using large-scale RNA interference (RNAi) screening, we find that KDM3A, a histone H3 lysine 9 (H3K9) mono- and di-demethylase, plays a pivotal role in anoikis induction. In attached breast epithelial cells, KDM3A expression is maintained at low levels by integrin signaling. Following detachment, integrin signaling is decreased resulting in increased KDM3A expression. RNAi-mediated knockdown of KDM3A substantially reduces apoptosis following detachment and, conversely, ectopic expression of KDM3A induces cell death in attached cells. We find that KDM3A promotes anoikis through transcriptional activation of BNIP3 and BNIP3L, which encode pro-apoptotic proteins. Using mouse models of breast cancer metastasis we show that knockdown of Kdm3a enhances metastatic potential. Finally, we find defective KDM3A expression in human breast cancer cell lines and tumors. Collectively, our results reveal a novel transcriptional regulatory program that mediates anoikis.

Aversa Z, Pin F, Lucia S, et al.
Autophagy is induced in the skeletal muscle of cachectic cancer patients.
Sci Rep. 2016; 6:30340 [PubMed] Free Access to Full Article Related Publications
Basal rates of autophagy can be markedly accelerated by environmental stresses. Recently, autophagy has been involved in cancer-induced muscle wasting. Aim of this study has been to evaluate if autophagy is induced in the skeletal muscle of cancer patients. The expression (mRNA and protein) of autophagic markers has been evaluated in intraoperative muscle biopsies. Beclin-1 protein levels were increased in cachectic cancer patients, suggesting autophagy induction. LC3B-I protein levels were not significantly modified. LC3B-II protein levels were significantly increased in cachectic cancer patients suggesting either increased autophagosome formation or reduced autophagosome turnover. Conversely, p62 protein levels were increased in cachectic and non-cachectic cancer patients, suggesting impaired autophagosome clearance. As for mitophagy, both Bnip3 and Nix/Bnip3L show a trend to increase in cachectic patients. In the same patients, Parkin levels significantly increased, while PINK1 was unchanged. At gene level, Beclin-1, p-62, BNIP3, NIX/BNIP3L and TFEB mRNAs were not significantly modulated, while LC3B and PINK1 mRNA levels were increased and decreased, respectively, in cachectic cancer patients. Autophagy is induced in the skeletal muscle of cachectic cancer patients, although autophagosome clearance appears to be impaired. Further studies should evaluate whether modulation of autophagy could represent a relevant therapeutic strategy in cancer cachexia.

Yang XJ, Si RH, Liang YH, et al.
Mir-30d increases intracellular survival of Helicobacter pylori through inhibition of autophagy pathway.
World J Gastroenterol. 2016; 22(15):3978-91 [PubMed] Free Access to Full Article Related Publications
AIM: To determine if mir-30d inhibits the autophagy response to Helicobacter pylori (H. pylori) invasion and increases H. pylori intracellular survival.
METHODS: The expression of mir-30d was detected by quantitative polymerase chain reaction (PCR), and autophagy level was examined by transmission electron microscopy, western blot, and GFP-LC3 puncta assay in human AGS cells and GES-1 cells. Luciferase reporter assay was applied to confirm the specificity of mir-30d regulation on the expression of several core molecules involved in autophagy pathway. The expression of multiple core proteins were analyzed at both the mRNA and protein level, and the intracellular survival of H. pylori after different treatments was detected by gentamicin protection assay.
RESULTS: Autophagy level was increased in AGS and GES-1 cells in response to H. pylori infection, which was accompanied by upregulation of mir-30d expression (P < 0.05, vs no H. pylori infection). In the two gastric epithelial cell lines, mimic mir-30d was found to repress the autophagy process, whereas mir-30d inhibitor increased autophagy response to H. pylori invasion. mir-30d mimic decreased the luciferase activity of wild type reporter plasmids carrying the 3' untranslated region (UTR) of all five tested genes (ATG2B, ATG5, ATG12, BECN1, and BNIP3L), whereas it had no effect on the mutant reporter plasmids. These five genes are core genes of autophagy pathway, and their expression was reduced significantly after mir-30d mimic transfection (P < 0.05, vs control cells without mir-30d mimic treatment). Mir-30d mimic transfection and direct inhibition of autophagy increased the intracellular survival of H. pylori in AGS cells.
CONCLUSION: Mir-30d increases intracellular survival of H. pylori in gastric epithelial cells through inhibition of multiple core proteins in the autophagy pathway.

Lin Z, Li JW, Wang Y, et al.
Abnormal miRNA-30e Expression is Associated with Breast Cancer Progression.
Clin Lab. 2016; 62(1-2):121-8 [PubMed] Related Publications
BACKGROUND: microRNAs (miRNAs) are involved in the regulation of various cellular processes, such as differentiation, proliferation, metabolism, and apoptosis, and they have been implicated in several diseases, including cancers.
METHODS: To assess the role of miRNA in the progression of breast cancer, we performed TaqMan-based miRNA profiling for plasma from patients with breast cancer (n = 53), unrelated diseases (n = 40), or matched healthy controls (n = 40), and for breast tumors or adjacent non-tumors (n = 41).
RESULTS: We selected 18 miRNAs with predicted roles in breast cancer and demonstrated that let-7i (p = 0.019), let-7a (p = 0.02), and miR-650 (p = 0.008) were significantly up-regulated in plasma; miR-21 (p < 0.001) is up-regulated in breast cancer tissue, and miR-30e was down-regulated in both plasma (p < 0.001) and breast cancer tissues (p = 0.004). Plasma miR-30e expression was shown to be statistically associated with age (p = 0.0402) and clinical stage (p = 0.007). However, receiver-operating characteristic curve analyses suggested that miR-30e expression cannot significantly differentiate breast cancer from healthy tissue or plasma. Consistent with a potential role for miR-30e in breast cancer, three predicted targets of miR-30e (RAB11A, BNIP3L, and RAB32) are up-regulated in breast cancer tissue.
CONCLUSIONS: These findings suggest that reduced miR-30e correlates with the clinical stage of breast cancer. It is worthwhile to further explore that the potential role of miR-30e as a tumor suppressor in breast cancer, as well as its potential therapeutic utility.

Vallée MP, Di Sera TL, Nix DA, et al.
Adding In Silico Assessment of Potential Splice Aberration to the Integrated Evaluation of BRCA Gene Unclassified Variants.
Hum Mutat. 2016; 37(7):627-39 [PubMed] Free Access to Full Article Related Publications
Clinical mutation screening of the cancer susceptibility genes BRCA1 and BRCA2 generates many unclassified variants (UVs). Most of these UVs are either rare missense substitutions or nucleotide substitutions near the splice junctions of the protein coding exons. Previously, we developed a quantitative method for evaluation of BRCA gene UVs-the "integrated evaluation"-that combines a sequence analysis-based prior probability of pathogenicity with patient and/or tumor observational data to arrive at a posterior probability of pathogenicity. One limitation of the sequence analysis-based prior has been that it evaluates UVs from the perspective of missense substitution severity but not probability to disrupt normal mRNA splicing. Here, we calibrated output from the splice-site fitness program MaxEntScan to generate spliceogenicity-based prior probabilities of pathogenicity for BRCA gene variants; these range from 0.97 for variants with high probability to damage a donor or acceptor to 0.02 for exonic variants that do not impact a splice junction and are unlikely to create a de novo donor. We created a database that provides the combined missense substitution severity and spliceogenicity-based probability of pathogenicity for BRCA gene single-nucleotide substitutions. We also updated the BRCA gene Ex-UV LOVD, available at, with 77 re-evaluable variants.

Demark-Wahnefried W, Nix JW, Hunter GR, et al.
Feasibility outcomes of a presurgical randomized controlled trial exploring the impact of caloric restriction and increased physical activity versus a wait-list control on tumor characteristics and circulating biomarkers in men electing prostatectomy for prostate cancer.
BMC Cancer. 2016; 16:61 [PubMed] Free Access to Full Article Related Publications
BACKGROUND: Obesity is associated with tumor aggressiveness and disease-specific mortality for more than 15 defined malignancies, including prostate cancer. Preclinical studies suggest that weight loss from caloric restriction and increased physical activity may suppress hormonal, energy-sensing, and inflammatory factors that drive neoplastic progression; however, exact mechanisms are yet to be determined, and experiments in humans are limited.
METHODS: We conducted a randomized controlled trial among 40 overweight or obese, newly-diagnosed prostate cancer patients who elected prostatectomy to explore feasibility of a presurgical weight loss intervention that promoted a weight loss of roughly one kg. week(-1) via caloric restriction and physical activity, as well as to assess effects on tumor biology and circulating biomarkers. Measures of feasibility (accrual, retention, adherence, and safety) were primary endpoints. Exploratory aims were directed at the intervention's effect on tumor proliferation (Ki-67) and other tumor markers (activated caspase-3, insulin and androgen receptors, VEGF, TNFβ, NFκB, and 4E-BP1), circulating biomarkers (PSA, insulin, glucose, VEGF, TNFβ, leptin, SHBG, and testosterone), lymphocytic gene expression of corresponding factors and cellular bioenergetics in neutrophils, and effects on the gut microbiome. Consenting patients were randomized in a 1:1 ratio to either: 1) weight loss via a healthful, guidelines-based diet and exercise regimen; or 2) a wait-list control. While biological testing is currently ongoing, this paper details our methods and feasibility outcomes.
RESULTS: The accrual target was met after screening 101 cases (enrollment rate: 39.6%). Other outcomes included a retention rate of 85%, excellent adherence (95%), and no serious reported adverse events. No significant differences by age, race, or weight status were noted between enrollees vs. non-enrollees. The most common reasons for non-participation were "too busy" (30%), medical exclusions (21%), and "distance" (16%).
CONCLUSIONS: Presurgical trials offer a means to study the impact of diet and exercise interventions directly on tumor tissue, and other host factors that are feasible and safe, though modifications are needed to conduct trials within an abbreviated period of time and via distance medicine-based approaches. Pre-surgical trials are critical to elucidate the impact of lifestyle interventions on specific mechanisms that mediate carcinogenesis and which can be used subsequently as therapeutic targets.

Zhao X, Cai H, Wang X, Ma L
Discovery of signature genes in gastric cancer associated with prognosis.
Neoplasma. 2016; 63(2):239-45 [PubMed] Related Publications
Gene expression profiles of gastric cancer (GC) were analyzed with bioinformatics tools to identify signature genes associated with prognosis. Four gene expression data sets (accession number: GSE2685, GSE30727, GSE38932 and GSE26253) were downloaded from Gene Expression Omnibus. Differentially expressed genes (DEGs) were screened out using significance analysis of microarrays (SAM) algorithm. P-value 1 were set as the threshold. A co-expression network was constructed for the GC-related genes with package WGCNA of R. Modules were disclosed with WGCNA algorithm. Survival-related signature genes were screened out via COX single-variable regression.A total of 3210 GC-related genes were identified from the 3 data sets. Significantly enriched GO biological process terms included cell death, cell proliferation, apoptosis, response to hormone and phosphorylation. Pathways like viral carcinogenesis, metabolism, EBV viral infection, and PI3K-AKT signaling pathway were significantly over-represented in the DEGs. A gene co-expression network including 2414 genes was constructed, from which 7 modules were revealed. A total of 17 genes were identified as signature genes, such as DAB2, ALDH2, CD58, CITED2, BNIP3L, SLC43A2, FAU and COL5A1.Many signature genes associated with prognosis of GC were identified in present study, some of which have been implicated in the pathogenesis of GC. These findings could not only improve the knowledge about GC, but also provide clues for clinical treatments.

Fader CM, Salassa BN, Grosso RA, et al.
Hemin induces mitophagy in a leukemic erythroblast cell line.
Biol Cell. 2016; 108(4):77-95 [PubMed] Related Publications
BACKGROUND INFORMATION: In eukaryotic cells, autophagy is considered a lysosomal catabolic process which participates in the degradation of intracellular components in a vacuolar structure termed autolysosome. This pathway plays a significant role in the erythropoiesis process, contributing to the clearance of some organelles (such as mitochondria) that are not necessary in the mature red blood cells. Nevertheless, the role of autophagy in erythrocyte maturation has not been fully established.
RESULTS: Here, we have demonstrated that hemin (a physiological erythroid maturation stimulator) is able to induce the expression of critical autophagic genes (i.e., Map1a1b (LC3), Beclin-1 gen, Atg5) in an erythroleukemia cell type. We have also shown that hemin increased the size of autophagic vacuoles which were labelled with LC3 and the degradative lysosomal marker dye quenched-bovine serum albumin. In addition, we have determined by Western blot a rise in the lipidated form of the autophagic protein LC3 (i.e., LC3-II) upon hemin treatment. Moreover, we provide evidence that hemin induces mitochondrial membrane depolarisation and that mitochondria sequestration by autophagy requires the active form of the NIX protein.
CONCLUSIONS: We have found that the physiological erythroid maturation stimulator hemin is able to induce mitophagy in K562 cells, and that the autophagy adaptor NIX is necessary for mitophagy progression. K562 cells have been used as a relevant model to determine the possible therapeutic role of new differentiating compounds.
SIGNIFICANCE: It has been proposed that autophagy induction is a feasible new therapeutic key in fighting cancer. Our results suggest that hemin is favoring erythroid maturation by inducing an autophagic response in K562 cells, being a possible therapeutic candidate that may help in the chronic myelogenous leukemia (CML) treatment.

Wilfinger N, Austin S, Scheiber-Mojdehkar B, et al.
Novel p53-dependent anticancer strategy by targeting iron signaling and BNIP3L-induced mitophagy.
Oncotarget. 2016; 7(2):1242-61 [PubMed] Free Access to Full Article Related Publications
This study identifies BNIP3L as the key regulator of p53-dependent cell death mechanism in colon cancer cells targeted by the novel gallium based anticancer drug, KP46. KP46 specifically accumulated into mitochondria where it caused p53-dependent morphological and functional damage impairing mitochondrial dynamics and bioenergetics. Furthermore, competing with iron for cellular uptake, KP46 lowered the intracellular labile iron pools and intracellular heme. Accordingly, p53 accumulated in the nucleus where it activated its transcriptional target BNIP3L, a BH3 only domain protein with functions in apoptosis and mitophagy. Upregulated BNIP3L sensitized the mitochondrial permeability transition and strongly induced PARKIN-mediated mitochondrial clearance and cellular vacuolization. Downregulation of BNIP3L entirely rescued cell viability caused by exposure of KP46 for 24 hours, confirming that early induced cell death was regulated by BNIP3L. Altogether, targeting BNIP3L in wild-type p53 colon cancer cells is a novel anticancer strategy activating iron depletion signaling and the mitophagy-related cell death pathway.

Shinderman-Maman E, Cohen K, Weingarten C, et al.
The thyroid hormone-αvβ3 integrin axis in ovarian cancer: regulation of gene transcription and MAPK-dependent proliferation.
Oncogene. 2016; 35(15):1977-87 [PubMed] Related Publications
Ovarian carcinoma is the fifth common cause of cancer death in women, despite advanced therapeutic approaches. αvβ3 integrin, a plasma membrane receptor, binds thyroid hormones (L-thyroxine, T4; 3,5,3'-triiodo-L-thyronine, T3) and is overexpressed in ovarian cancer. We have demonstrated selective binding of fluorescently labeled hormones to αvβ3-positive ovarian cancer cells but not to integrin-negative cells. Physiologically relevant T3 (1 nM) and T4 (100 nM) concentrations in OVCAR-3 (high αvβ3) and A2780 (low αvβ3) cells promoted αv and β3 transcription in association with basal integrin levels. This transcription was effectively blocked by RGD (Arg-Gly-Asp) peptide and neutralizing αvβ3 antibodies, excluding T3-induced β3 messenger RNA, suggesting subspecialization of T3 and T4 binding to the integrin receptor pocket. We have provided support for extracellular regulated kinase (ERK)-mediated transcriptional regulation of the αv monomer by T3 and of β3 monomer by both hormones and documented a rapid (30-120 min) and dose-dependent (0.1-1000 nM) ERK activation. OVCAR-3 cells and αvβ3-deficient HEK293 cells treated with αvβ3 blockers confirmed the requirement for an intact thyroid hormone-integrin interaction in ERK activation. In addition, novel data indicated that T4, but not T3, controls integrin's outside-in signaling by phosphorylating tyrosine 759 in the β3 subunit. Both hormones induced cell proliferation (cell counts), survival (Annexin-PI), viability (WST-1) and significantly reduced the expression of genes that inhibit cell cycle (p21, p16), promote mitochondrial apoptosis (Nix, PUMA) and tumor suppression (GDF-15, IGFBP-6), particularly in cells with high integrin expression. At last, we have confirmed that hypothyroid environment attenuated ovarian cancer growth using a novel experimental platform that exploited paired euthyroid and severe hypothyroid serum samples from human subjects. To conclude, our data define a critical role for thyroid hormones as potent αvβ3-ligands, driving ovarian cancer cell proliferation and suggest that disruption of this axis may present a novel treatment strategy in this aggressive disease.

Jin Z, Zheng L, Xin X, et al.
Upregulation of forkhead box O3 transcription is involved in C2-ceramide induced apoptosis and autophagy in ovarian cancer cells in vitro.
Mol Med Rep. 2014; 10(6):3099-105 [PubMed] Related Publications
Ceramide is a bioactive lipid which functions as a tumor suppressor, mediating processes such as apoptosis, growth arrest, senescence and differentiation. The effects of ceramide in ovarian cancers have not been well established. The objective of the present study was to investigate the effects of C2‑ceramide treatment in A2780 ovarian cancer cells and its possible molecular mechanism. C2‑ceramide-induced proliferation inhibition was analyzed using an MTT assay and Trypan blue test. Flow cytometry and terminal deoxynucleotidyl transferase dUTP nick end labeling were used to identify the induction of apoptosis. Transmission electron microscopy was used to confirm the formation of autophagosomes. Quantitative polymerase chain reaction was performed to analyze the messenger RNA expression of the autophagy and cell death associated genes and western blotting was used to analyze the protein expression of beclin 1, LC3, Akt, forkhead box O3 (FOXO3) and adenosine monophosphate-activated protein kinase in ovarian cancer cells. It was found that C2‑ceramide inhibited A2780 cell proliferation in a time‑ and dose‑dependent manner and C2‑ceremide induced A2780 cell apoptosis and autophagy. However, C2‑ceramide‑induced autophagy did not result in cell death, but instead protected ovarian cancer cells from apoptosis. Akt inhibition and FOXO3 activation were implicated in C2‑ceramide‑treated ovarian cancer cells. Furthermore, FOXO3 target genes, which were associated with autophagy (MAP1LC3, GABARAP and GABARAPL1) and cell death (BNIP3, BNIP3L, BIM and PUMA), were upregulated. The present study has shown that C2‑ceramide induced apoptosis and autophagy in ovarian cancer cells. FOXO3 transcription was upregulated, which may contribute to C2‑ceramide‑induced apoptosis and autophagy.

Mirzaei MR, Najafi A, Arababadi MK, et al.
Altered expression of apoptotic genes in response to OCT4B1 suppression in human tumor cell lines.
Tumour Biol. 2014; 35(10):9999-10009 [PubMed] Related Publications
OCT4B1 is a newly discovered spliced variant of OCT4 which is primarily expressed in pluripotent and tumor cells. Based on our previous studies, OCT4B1 is significantly overexpressed in tumors, where it endows an anti-apoptotic property to tumor cells. However, the mechanism by which OCT4B1 regulates the apoptotic pathway is not yet elucidated. Here, we investigated the effects of OCT4B1 suppression on the expression alteration of 84 genes involved in apoptotic pathway. The AGS (gastric adenocarcinoma), 5637 (bladder tumor), and U-87MG (brain tumor) cell lines were transfected with OCT4B1 or irrelevant siRNAs. The expression level of apoptotic genes was then quantified using a human apoptosis panel-PCR kit. Our data revealed an almost similar pattern of alteration in the expression profile of apoptotic genes in all three studied cell lines, following OCT4B1 suppression. In general, the expression of more than 54 apoptotic genes (64 % of arrayed genes) showed significant changes. Among these, some up-regulated (CIDEA, CIDEB, TNFRSF1A, TNFRSF21, TNFRSF11B, TNFRSF10B, and CASP7) and down-regulated (BCL2, BCL2L11, TP73, TP53, BAD, TRAF3, TRAF2, BRAF, BNIP3L, BFAR, and BAX) genes had on average more than tenfold gene expression alteration in all three examined cell lines. With some minor exceptions, suppression of OCT4B1 caused upregulation of pro-apoptotic and down-regulation of anti-apoptotic genes in transfected tumor cells. Uncovering OCT4B1 down-stream targets could further elucidate its part in tumorigenesis, and could lead to finding a new approach to combat cancer, based on targeting OCT4B1.

Foulks JM, Carpenter KJ, Luo B, et al.
A small-molecule inhibitor of PIM kinases as a potential treatment for urothelial carcinomas.
Neoplasia. 2014; 16(5):403-12 [PubMed] Free Access to Full Article Related Publications
The proto-oncogene proviral integration site for moloney murine leukemia virus (PIM) kinases (PIM-1, PIM-2, and PIM-3) are serine/threonine kinases that are involved in a number of signaling pathways important to cancer cells. PIM kinases act in downstream effector functions as inhibitors of apoptosis and as positive regulators of G1-S phase progression through the cell cycle. PIM kinases are upregulated in multiple cancer indications, including lymphoma, leukemia, multiple myeloma, and prostate, gastric, and head and neck cancers. Overexpression of one or more PIM family members in patient tumors frequently correlates with poor prognosis. The aim of this investigation was to evaluate PIM expression in low- and high-grade urothelial carcinoma and to assess the role PIM function in disease progression and their potential to serve as molecular targets for therapy. One hundred thirty-seven cases of urothelial carcinoma were included in this study of surgical biopsy and resection specimens. High levels of expression of all three PIM family members were observed in both noninvasive and invasive urothelial carcinomas. The second-generation PIM inhibitor, TP-3654, displays submicromolar activity in pharmacodynamic biomarker modulation, cell proliferation studies, and colony formation assays using the UM-UC-3 bladder cancer cell line. TP-3654 displays favorable human ether-à-go-go-related gene and cytochrome P450 inhibition profiles compared with the first-generation PIM inhibitor, SGI-1776, and exhibits oral bioavailability. In vivo xenograft studies using a bladder cancer cell line show that PIM kinase inhibition can reduce tumor growth, suggesting that PIM kinase inhibitors may be active in human urothelial carcinomas.

Yang C, Jiang L, Zhang H, et al.
Analysis of hypoxia-induced metabolic reprogramming.
Methods Enzymol. 2014; 542:425-55 [PubMed] Related Publications
Hypoxia is a common finding in advanced human tumors and is often associated with metastatic dissemination and poor prognosis. Cancer cells adapt to hypoxia by utilizing physiological adaptation pathways that promote a switch from oxidative to glycolytic metabolism. This promotes the conversion of glucose into lactate while limiting its transformation into acetyl coenzyme A (acetyl-CoA). The uptake of glucose and the glycolytic flux are increased under hypoxic conditions, mostly owing to the upregulation of genes encoding glucose transporters and glycolytic enzymes, a process that depends on hypoxia-inducible factor 1 (HIF-1). The reduced delivery of acetyl-CoA to the tricarboxylic acid cycle leads to a switch from glucose to glutamine as the major substrate for fatty acid synthesis in hypoxic cells. In addition, hypoxia induces (1) the HIF-1-dependent expression of BCL2/adenovirus E1B 19-kDa interacting protein 3 (BNIP3) and BNIP3-like (BNIP3L), which trigger mitochondrial autophagy, thereby decreasing the oxidative metabolism of both fatty acids and glucose, and (2) the expression of the sodium-hydrogen exchanger NHE1, which maintains an alkaline intracellular pH. Here, we present a compendium of methods to study hypoxia-induced metabolic alterations.

Chuang WY, Ströbel P, Bohlender-Willke AL, et al.
Late-onset myasthenia gravis - CTLA4(low) genotype association and low-for-age thymic output of naïve T cells.
J Autoimmun. 2014; 52:122-9 [PubMed] Related Publications
Late-onset myasthenia gravis (LOMG) has become the largest MG subgroup, but the underlying pathogenetic mechanisms remain mysterious. Among the few etiological clues are the almost unique serologic parallels between LOMG and thymoma-associated MG (TAMG), notably autoantibodies against acetylcholine receptors, titin, ryanodine receptor, type I interferons or IL-12. This is why we checked LOMG patients for two further peculiar features of TAMG - its associations with the CTLA4(high/gain-of-function) +49A/A genotype and with increased thymic export of naïve T cells into the blood, possibly after defective negative selection in AIRE-deficient thymomas. We analyzed genomic DNA from 116 Caucasian LOMG patients for CTLA4 alleles by PCR/restriction fragment length polymorphism, and blood mononuclear cells for recent thymic emigrants by quantitative PCR for T cell receptor excision circles. In sharp contrast with TAMG, we now find that: i) CTLA4(low) +49G(+) genotypes were more frequent (p = 0.0029) among the 69 LOMG patients with age at onset ≥60 years compared with 172 healthy controls; ii) thymic export of naïve T cells from the non-neoplastic thymuses of 36 LOMG patients was lower (p = 0.0058) at diagnosis than in 77 age-matched controls. These new findings are important because they suggest distinct initiating mechanisms in TAMG and LOMG and hint at aberrant immuno-regulation in the periphery in LOMG. We therefore propose alternate defects in central thymic or peripheral tolerance induction in TAMG and LOMG converging on similar final outcomes. In addition, our data support a 60-year-threshold for onset of 'true LOMG' and an LOMG/early-onset MG overlapping group of patients between 40 and 60.

Pereira-Caro G, Mateos R, Traka MH, et al.
Hydroxytyrosyl ethyl ether exhibits stronger intestinal anticarcinogenic potency and effects on transcript profiles compared to hydroxytyrosol.
Food Chem. 2013; 138(2-3):1172-82 [PubMed] Related Publications
The anticarcinogenic activity of hydroxytyrosyl ethyl ether (HTy-Et) compared to its precursor hydroxytyrosol (HTy) has been studied in human Caco-2 colon adenocarcinoma cells. 451 and 977 genes were differentially expressed in Caco-2 cells exposed to HTy or HTy-Et for 24h, respectively, compared with untreated cells (P<0.005; FDR=0), using Affymetrix microarrays. Results showed that both HTy and HTy-Et inhibited cell proliferation and arrested the cell cycle by up-regulating p21 and CCNG2 and down-regulating CCNB1 protein expression. HTy and HTy-Et also altered the transcription of specific genes involved in apoptosis, as suggested by the up-regulation of BNIP3, BNIP3L, PDCD4 and ATF3 and the activation of caspase-3. Moreover, these polyphenols up-regulated xenobiotic metabolizing enzymes UGT1A10 and CYP1A1, enhancing carcinogen detoxification. In conclusion, these results highlight that HTy and its derivative HTy-Et modulate molecular mechanisms involved in colon cancer, with HTy-Et being more effective than HTy.

Yang X, Zhong X, Tanyi JL, et al.
mir-30d Regulates multiple genes in the autophagy pathway and impairs autophagy process in human cancer cells.
Biochem Biophys Res Commun. 2013; 431(3):617-22 [PubMed] Free Access to Full Article Related Publications
In human epithelial cancers, the microRNA (miRNA) mir-30d is amplified with high frequency and serves as a critical oncomir by regulating metastasis, apoptosis, proliferation, and differentiation. Autophagy, a degradation pathway for long-lived protein and organelles, regulates the survival and death of many cell types. Increasing evidence suggests that autophagy plays an important function in epithelial tumor initiation and progression. Using a combined bioinformatics approach, gene set enrichment analysis, and miRNA target prediction, we found that mir-30d might regulate multiple genes in the autophagy pathway including BECN1, BNIP3L, ATG12, ATG5, and ATG2. Our further functional experiments demonstrated that the expression of these core proteins in the autophagy pathway was directly suppressed by mir-30d in cancer cells. Finally, we showed that mir-30d regulated the autophagy process by inhibiting autophagosome formation and LC3B-I conversion to LC3B-II. Taken together, our results provide evidence that the oncomir mir-30d impairs the autophagy process by targeting multiple genes in the autophagy pathway. This result will contribute to understanding the molecular mechanism of mir-30d in tumorigenesis and developing novel cancer therapy strategy.

Lu Y, Wang L, He M, et al.
Nix protein positively regulates NF-κB activation in gliomas.
PLoS One. 2012; 7(9):e44559 [PubMed] Free Access to Full Article Related Publications
Previous reports indicate that the NIX/BNIP3L gene acts as a pro-apoptotic factor by interacting with BCL2 and BCL-XL, playing an important role in hypoxia-dependent cell death and acting as a tumor suppressor. However, many studies also showed that NIX is linked to a protective role and cell survival in cancer cells. Nuclear factor-κB (NF-κB) can attenuate apoptosis in human cancers in response to chemotherapeutic agents and ionizing radiation. We observed an absence of i-κBα (NF-κB activation inhibitor) expression, but a greater expression of Nix and p-NF-κB proteins in the Nix-wt U251 cells, which was not observed in the Nix-kn cells under hypoxic conditions. Using electrophoretic mobility shift assay (EMSA) and luciferase detection, the activation of NF-κB was detected only in the Nix-wt U251 cells with hypoxia. These data imply that Nix protein might play a role in the positive regulation of the NF-κB pathway. Moreover, 46 cases of glioma also showed high levels of Nix protein expression, which was always accompanied by high p-NF-κB expression. Patients with Nix (+) showed less tissue apoptosis behavior in glioblastoma (GBM), unlike that observed in the Nix-negative patients (-). The same apoptotic tendency was also identified in anaplastic astrocytoma (AA) groups, but not in astrocytoma (AS). On analyzing the Kaplan-Meier curve, better tumor-free survival was observed only in cases of astrocytoma, and not in AA and GBM. Thus, our study indicates that Nix protein might have multiple functions in regulating glioma behaviors. In the low-grade gliomas (astrocytoma) with low expression of NF-κB, the cell death-inducing function that occurs through a Bax mechanism might predominate and act as a tumor suppressor. While in the malignant gliomas (AA and GBM), with higher expression of the NIX gene and with activity of the NF-κB pathway, the oncogene function of Nix was predominant.

Jin X, Wu XX, Jin C, et al.
Delineation of apoptotic genes for synergistic apoptosis of lexatumumab and anthracyclines in human renal cell carcinoma cells by polymerase chain reaction array.
Anticancer Drugs. 2012; 23(4):445-54 [PubMed] Related Publications
Lexatumumab, a human agonistic monoclonal antibody against tumor necrosis factor (TNF)-related apoptosis-inducing ligand receptor-2 (TRAIL-R2), is a promising molecular-targeted therapeutic agent. Our past study indicated that low concentrations of doxorubicin sensitized renal cell carcinoma (RCC) cells to lexatumumab-mediated apoptosis. The present study was designed to examine the cellular and molecular effects of lexatumumab and anthracyclines in RCC cells. The treatment of human RCC cells with lexatumumab in combination with anthracyclines, epirubicin, and pirarubicin had a synergistic cytotoxicity. A marked synergistic apoptosis was induced by lexatumumab in combination with epirubicin or pirarubicin. Epirubicin and pirarubicin significantly increased the TRAIL-R2 expression at both the mRNA and the protein levels. The combination-induced cytotoxicity was significantly suppressed by the human recombinant DR5:Fc chimeric protein. To further explore the molecular mechanisms in this synergistic cytotoxicity with lexatumumab and anthracyclines, the changes in 84 apoptosis-related genes were evaluated by a quantitative polymerase chain reaction (PCR) array. Among these genes, 18 (CD40LG, FASLG, LTA, TNSF7, FAS, BAG3, BAK1, BAX, BID, BIK, BCL10, caspase-1, caspase-5, caspase-6, caspase-10, TNF receptor-associated factor 1, PYCARD, and CIDEA) were significantly upregulated and eight (TNF receptor-associated factor 4, TNFRSF11B, TNF, BCL2, BCL2L1, BNIP3L, caspase-9, and DAPK1) were downregulated at mRNA levels in RCC cells cotreated with lexatumumab and epirubicin. Furthermore, the upregulation of mRNA levels of PYCARD and CIDEA was confirmed using real-time reverse transcriptase-PCR analysis. The present study demonstrates that anthracylines sensitize RCC cells to lexatumumab-mediated apoptosis by inducing TRAIL-R2 expression, and the utility of PCR array to elucidate the mechanism of synergistic apoptosis.

Knutson AK, Welsh J, Taylor T, et al.
Comparative effects of histone deacetylase inhibitors on p53 target gene expression, cell cycle and apoptosis in MCF-7 breast cancer cells.
Oncol Rep. 2012; 27(3):849-53 [PubMed] Related Publications
Histone deacetylase inhibitors are currently being evaluated for their therapeutic potential and have shown considerable promise as adjuvant therapies for a number of cancers. This study compared the effects of 2 hydroxamic acid based inhibitors, CG-1521 and SAHA, on gene expression, cell cycle and cell death in MCF-7 human breast cancer cells. Both compounds show a dose- and time-dependent effect on cell number (evaluated using crystal violet), however CG-1521 exerts its effects significantly earlier than SAHA, and CG-1521 induces apoptosis (assessed by Apo-BrdU staining and flow cytometry) more rapidly than SAHA. qPCR of cell cycle regulatory and apoptotic genes shows that CG-1521 and SAHA modulate similar cohorts of p53-responsive genes, however, the levels of induction and the timing of the induction differs significantly between the 2 inhibitors. In particular SAHA downregulates cell cycle-associated genes that modulate the G1/S transition (including cyclin D1 and cdc25a) and the G2/M transition [cyclin B1, Plk1, Stk6 (serine-threonine kinase 6, Aurora kinase A) and Kntc2] more significantly than CG-1521. In contrast, CG-1521 significantly induces the expression of several p53 target genes associated with apoptosis including Bnip3/Bnip3L, p21/p21B and Gdf15. The differential levels of gene induction provide molecular evidence of both cell cycle arrest and apoptosis, and suggest a molecular mechanism that explains the difference in the biological effects of the 2 histone deacetylase inhibitors.

Whitaker-Menezes D, Martinez-Outschoorn UE, Flomenberg N, et al.
Hyperactivation of oxidative mitochondrial metabolism in epithelial cancer cells in situ: visualizing the therapeutic effects of metformin in tumor tissue.
Cell Cycle. 2011; 10(23):4047-64 [PubMed] Free Access to Full Article Related Publications
We have recently proposed a new mechanism for explaining energy transfer in cancer metabolism. In this scenario, cancer cells behave as metabolic parasites, by extracting nutrients from normal host cells, such as fibroblasts, via the secretion of hydrogen peroxide as the initial trigger. Oxidative stress in the tumor microenvironment then leads to autophagy-driven catabolism, mitochondrial dys-function, and aerobic glycolysis. This, in turn, produces high-energy nutrients (such as L-lactate, ketones, and glutamine) that drive the anabolic growth of tumor cells, via oxidative mitochondrial metabolism. A logical prediction of this new "parasitic" cancer model is that tumor-associated fibroblasts should show evidence of mitochondrial dys-function (mitophagy and aerobic glycolysis). In contrast, epithelial cancer cells should increase their oxidative mitochondrial capacity. To further test this hypothesis, here we subjected frozen sections from human breast tumors to a staining procedure that only detects functional mitochondria. This method detects the in situ enzymatic activity of cytochrome C oxidase (COX), also known as Complex IV. Remarkably, cancer cells show an over-abundance of COX activity, while adjacent stromal cells remain essentially negative. Adjacent normal ductal epithelial cells also show little or no COX activity, relative to epithelial cancer cells. Thus, oxidative mitochondrial activity is selectively amplified in cancer cells. Although COX activity staining has never been applied to cancer tissues, it could now be used routinely to distinguish cancer cells from normal cells, and to establish negative margins during cancer surgery. Similar results were obtained with NADH activity staining, which measures Complex I activity, and succinate dehydrogenase (SDH) activity staining, which measures Complex II activity. COX and NADH activities were blocked by electron transport inhibitors, such as Metformin. This has mechanistic and clinical implications for using Metformin as an anti-cancer drug, both for cancer therapy and chemo-prevention. We also immuno-stained human breast cancers for a series of well-established protein biomarkers of metabolism. More specifically, we now show that cancer-associated fibroblasts over-express markers of autophagy (cathepsin B), mitophagy (BNIP3L), and aerobic glycolysis (MCT4). Conversely, epithelial cancer cells show the over-expression of a mitochondrial membrane marker (TOMM20), as well as key components of Complex IV (MT-CO1) and Complex II (SDH-B). We also validated our observations using a bioinformatics approach with data from > 2,000 breast cancer patients, which showed the transcriptional upregulation of mitochondrial oxidative phosphorylation (OXPHOS) in human breast tumors (p < 10(-20)), and a specific association with metastasis. Therefore, upregulation of OXPHOS in epithelial tumor cells is a common feature of human breast cancers. In summary, our data provide the first functional in vivo evidence that epithelial cancer cells perform enhanced mitochondrial oxidative phosphorylation, allowing them to produce high amounts of ATP. Thus, we believe that mitochondria are both the "powerhouse" and "Achilles' heel" of cancer cells.

Munksgaard Persson M, Johansson ME, Monsef N, et al.
HIF-2α expression is suppressed in SCLC cells, which survive in moderate and severe hypoxia when HIF-1α is repressed.
Am J Pathol. 2012; 180(2):494-504 [PubMed] Related Publications
Small cell lung carcinoma (SCLC) is extremely aggressive and frequently metastasizes widely in its early stage. Because tumor hypoxia is related to aggressive tumor behavior and the hypoxic adaptation of SCLC is poorly documented, we stained SCLC tumors arranged in a tissue microarray for hypoxia-inducible factor (HIF)-1α and HIF-2α proteins. We found an overall lack of HIF-2α protein expression, which was confirmed in large tumor sections. HIF-1α protein was strongly expressed in most tumors, frequently adjacent to necrotic regions. In concordance, cultured SCLC but not non-small cell lung carcinoma cells showed no or extremely low levels of HIF-2α mRNA and no HIF-2α protein at hypoxia. HIF-1α was stabilized after 4 hours at hypoxia, and its accumulation increased up to 96 hours. SCLC cells survived well and showed net proliferation and low cell death in modest (1% oxygen) and severe (0.1% oxygen) hypoxia. HIF-1α repression virtually did not influence cell death or viability despite reduced levels of hypoxia-inducible genes, such as BNIP3 and BNIP3L. At 1% oxygen no increased autophagy (LC3B-II activation) or NF-κB signaling were detected, whereas the unfolded protein response was activated at severe hypoxia. Our data indicate that HIFs are not exclusively required for SCLC cell survival at modest or severe hypoxia and that additional, yet uncharacterized, hypoxia-driven adaptation pathways may become activated.

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