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Increased sensitivity to glucose starvation correlates with downregulation of glycogen phosphorylase isoform PYGB in tumor cell lines resistant to 2-deoxy-d-glucose

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Abstract

Background

As tumors evolve, they upregulate glucose metabolism while also encountering intermittent periods of glucose deprivation. Here, we investigate mechanisms by which pancreatic cancer cells respond to therapeutic (2-deoxy-d-glucose, 2-DG) and physiologic (glucose starvation, GS) forms of glucose restriction.

Methods

From a tumor cell line (1420) that is unusually sensitive to 2-DG under normoxia, low (14DG2)- and high (14DG5)-dose resistant cell lines were selected and used to probe the metabolic pathways involved with their response to different forms of glucose deprivation.

Results

Muted induction of the unfolded protein response was found to correlate with resistance to 2-DG. Additionally, 14DG2 displayed reduced 2-DG uptake, while 14DG5 was cross-resistant to tunicamycin, suggesting it has enhanced ability to manage glycosylation defects. Conversely, 2-DG-resistant cell lines were more sensitive than their parental cell line to GS, which coincided with lowered levels of glycogen phosphorylase (PYGB) and reduced breakdown of glycogen to glucose in the 2-DG-resistant cell lines. Moreover, by inhibiting PYGB in the parental cell line, sensitivity to GS was increased.

Conclusions

Overall, the data demonstrate that the manner in which glucose is restricted in tumor cells, i.e., therapeutic or physiologic, leads to differential biological responses involving distinct glucose metabolic pathways. Moreover, in evolving tumors where glucose restriction occurs, the identification of PYGB as a metabolic target may have clinical application.

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Abbreviations

2-DG:

2-Deoxy-d-glucose

GS:

Glucose starvation

UPR:

Unfolded protein response

LLO:

Lipid-linked oligosaccharide

ER:

Endoplasmic reticulum

Grp78:

Glucose-regulated protein 78 kDa

PERK:

PKR-like ER kinase

CHOP:

C/EBP-homologous protein

LC3B:

Autophagy marker light chain 3

FACE:

Fluorophore-assisted carbohydrate electrophoresis

GYS:

Glycogen synthase 1

PYGB:

Glycogen phosphorylase brain isoform

TM:

Tunicamycin

BFA:

Brefeldin A

AMPK:

AMP-activated protein kinase

CaMKKβ:

Ca2+/calmodulin-dependent protein kinase kinase-beta

ROS:

Reactive oxygen species

ERK:

Extracellular signal-regulated kinases

SREBF1:

Sterol regulatory element-binding transcription factor 1

SREBF2:

Sterol regulatory element-binding transcription factor 2

INSIG1:

Insulin-induced gene 1

INSIG2:

Insulin-induced gene 2

SCAP:

SREBF chaperone

MBTPS1:

Membrane-bound transcription factor peptidase site 1

MBTPS2:

Membrane-bound transcription factor peptidase site 2

Rapa:

Rapamycin

3-MA:

3-Methyladenine

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Acknowledgments

We would like to thank Dr. Otto Baba (Department of Hard Tissue Engineering, Tokyo Medical and Dental University, Tokyo, Japan) for kindly providing the anti-glycogen antibody. We are grateful to the following persons from University of Miami for their technical support on the following: Dr. Eli Gilboa and Dr. Mansoor Ahmed for radioactive uptake assays, Dr. Enrique Mesri for qPCR and David Siefker for fluorescent microscopy. We also want to acknowledge the following for thoughtful discussions and insights on this work: Dr. Niramol Savaraj (University of Miami), Dr. Medhi Wangpaichitr (University of Miami) and Dr. Haibin Xi (University of California Los Angeles). This work was supported by National Cancer Institute grant CA37109 and Pap Corps award to TJL and National Institute of General Medical Sciences grant GM38545 to MAL.

Author information

Authors and Affiliations

  1. Sheila and David Fuente Graduate Program in Cancer Biology, University of Miami Miller School of Medicine, 1550 NW 10th Avenue, Fox Building #406, Miami, FL, 33136, USA

    Katherine B. Philips & Howard J. Leung

  2. Sylvester Comprehensive Cancer Center, University of Miami Miller School of Medicine, PAP Building, Room 115, 1550 NW 10th Ave, Miami, FL, 33136, USA

    Metin Kurtoglu & Huaping Liu

  3. Department of Pharmacology, University of Texas Southwestern Medical Center, 6001 Forest Park Rd., Dallas, TX, 75390-9041, USA

    Ningguo Gao & Mark A. Lehrman

  4. Department of Ophthalmology, Bascom Palmer Eye Institute, University of Miami, 900 NW 17th St., Miami, FL, 33136, USA

    Timothy G. Murray

  5. Department of Cell Biology (R-124), University of Miami Miller School of Medicine, 1600 NW 10th Avenue, Rosenstiel Medical Sciences Building #4026/4027, P.O. Box 016960, Miami, FL, 33101, USA

    Theodore J. Lampidis

  6. Sylvester Comprehensive Cancer Center, University of Miami Miller School of Medicine, 1475 NW 12th Avenue, P.O. Box 016960, Miami, FL, 33101, USA

    Theodore J. Lampidis

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  1. Katherine B. Philips
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  2. Metin Kurtoglu
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Correspondence to Theodore J. Lampidis.

Electronic supplementary material

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280_2013_2358_MOESM1_ESM.ppt

Figure S1. Autophagy induction does not correlate with resistance to 2-DG. a 1420, 14DG2 and 14DG5 cells were treated with the indicated doses of 2-DG for 24 h under normoxia then harvested and immunoblotting was performed to detect protein levels of Beclin and LC3B cleavage in the four cell lines. β-Actin was used as a loading control. b Cells were treated with the indicated doses of 3-MA and 2-DG for 72 h in normoxia and percent dead cells was assayed by trypan blue exclusion. The bars represent the average of triplicate samples ± SD. c Cell lines were treated with the indicated doses of rapamycin and 2-DG for 72 h in normoxia and percent dead cells was assayed by trypan blue exclusion. The bars represent the average of triplicate samples ± SD (PPT 231 kb)

280_2013_2358_MOESM2_ESM.ppt

Figure S2. Resistant cell lines not maintained with 2-DG display unique phenotypes. a Cell lines were treated with the indicated doses of 2-DG for 72 h in normoxia and percent dead cells was assayed by trypan blue exclusion. The bars represent the average of triplicate samples ± SD. ** P < 0.01, *** P < 0.001 as compared with 14DG2. b 1420, 14DG2 and 14DG5 cell lines were challenged with GS for 72 h in normoxia and percent dead cells was assayed by trypan blue exclusion. The bars represent the average of triplicate samples ± SD. * P < 0.05, ** P < 0.01, 14DG2NM is compared to 14DG2; 14DG5NM is compared to 14DG5. c Immunoblotting was performed to detect basal protein levels of PYGB in the four cell lines. β-Actin was used as a loading control. Quantification can be seen at right of blot (PPT 200 kb)

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Philips, K.B., Kurtoglu, M., Leung, H.J. et al. Increased sensitivity to glucose starvation correlates with downregulation of glycogen phosphorylase isoform PYGB in tumor cell lines resistant to 2-deoxy-d-glucose. Cancer Chemother Pharmacol 73, 349–361 (2014). https://doi.org/10.1007/s00280-013-2358-8

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  • DOI: https://doi.org/10.1007/s00280-013-2358-8

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