Summer Undergraduate Research Program - Fred Hutchinson ...

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Nancy Liu1 , Fionnuala Morrish2 , and David Hockenbery2 Metabolic Flexibility in Triple Negative Breast Cancer Cell Lines
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1 Swarthmore College, Swarthmore, PA; 2 Fred Hutchinson Cancer Research Center, Seattle, WA
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miR375 ANALYSIS LACTATE FLUX
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Lactate Dehydrogenase Assay
In Silico method of analysis
STAT3 EXPRESSION
Method: Assay for STAT3 and p-STAT3 in MCF7 (receptor positive) and
BT549 (triple negative) breast cancer cell lines
Cells lysed and protein quantified
Coomassie stain for protein verification
Tubulin loading control
Western analysis and protein detection
INTRODUCTION
Triple negative breast cancer cell lines have no estrogen receptor (ER),
progesterone receptor (PR) or human epidermal growth factor receptor 2
(HER2) compared to receptor positive cell lines. Triple negative breast
cancers often have limited treatment options since hormone therapies that
target these receptors are ineffective. Exploiting the metabolic differences
in triple negative breast cancer cell lines may elucidate new possible
targets for therapy.
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2012 Best Poster Award
Expression of STAT3
BT549 MCF7
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Central Carbon Metabolism
nted poster call
.649.3004
Method: To find the predicted and validated targets of miR375 and the
associated pathways, in silico analysis tools were used, including
miRWalk, GSEA, protein strings, and NCI-60 z-score analysis data.
ers
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BT549 MCF7
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Image courtesy of Fionnuala Morrish
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BT474
lactate
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BT474
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BT474
lactate
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Control BT474
(80ug)
Past studies have found that triple negative breast cancer cells have a
unique metabolic profile and a low OCR/ECAR ratio compared to receptor
positive cancer cell lines. This phenotype is related to the expression of
genes in central carbon metabolism, such as lactate dehydrogenase
(LDHA, LDHB), pyruvate kinase (PDK1), and the monocarboxylic acid
transporter (SLC16A1). Potential candidates for the transcriptional
regulation of these genes include STAT3 and miR375.
Method: Treat BT549 and BT474 (receptor positive) cell lines with lactate
and observe metabolic reprogramming with lactate dehydrogenase activity.
Results: BT474 cell lines may increase expression of skeletal LDH after
Results: STAT3 is potentially upregulated in BT549 cell lines compared to
MCF7 cell lines. Levels of p-STAT3 require 20ug-40ug of protein to be
detected (not shown).
lactate treatment.
older to the
DISCUSSION
Results: miR375 regulates many pathways that may explain the differing
bioenergetic phenotype between triple negative and receptor positive breast
cancer cells. Many putative metabolic pathways have not been studied and
should be exploited in the future.
miR375 EXPRESSION
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Differential expression of STAT3 and miR375 between receptor positive
and triple negative cell lines suggest that they play a role in the unique
metabolic phenotype of triple negative cells.
PLAG1
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All results were preliminary and replicates should be conducted, as well as
an expansion of the experiments on different receptor positive or triple
negative cell lines. Possible further experiments should determine the
mechanism of the lactate metabolic switch as well as whether giving a
lactate holiday will be able to reset this switch. Experiments
overexpressing miR375 in triple negative breast cancer cell lines will
explore results of the in silico miR375 analysis and elucidate the role of
miR375 in metabolic reprogramming.
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Image courtesy of Fionnuala Morrish
ACKNOWLEDGEMENTS
Method: Analysis of CellMiner miRNA data to determine differential
expression of miR375 in receptor positive (MCF7, T47D) with triple
negative (HS578T, BT549, MDAMB231) cell lines.
OBJECTIVES
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Thank you to Fionnuala Morrish, Kusum Chawla, Cori Abicoff, Li Huang,
Daciana Margienteau, and David Hockenbery for hosting me this summer
and for their guidance and support.
Result: miR375 is down regulated in all triple negative cancer cell lines
analyzed compared to receptor positive lines. This data has been confirmed
by an independent analysis of OSU and Agilent Human miRNA V2
(NCI60 data analysis tools, Reference: Reinhold WC, Sunshine M, Liu H,
Varma S, Kohn KW, Morris J, Doroshow J, and Pommier Y. 2012)
To determine the phosphorylation of STAT3 in receptor positive and triple
negative breast cancer cell lines.
Funding for this project was provided by Seattle Cancer Consortium Breast
SPORE NIH/NCI P50 CA 138293-02. The Summer Undergraduate
Research Program is supported in part by the Cancer Center Support Grant
(CCSG) CURE Supplement: 3 P30 CA015704-38S1.
To perform an in silico analysis of miR375 expression in receptor positive
and triple negative breast cancer cell lines and predict the consequences of
downregulating miR375 on downstream signaling and metabolic pathways.
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PLAG1 is a transcription factor differentially up regulated in triple
negative cells and acts as a proto-oncogene. Fatty acid combustion and
glucose utilization pathways are also associated with miR375 expression.
Reference: CellMiner: A Web-Based Suite of Genomic and Pharmacologic
Tools to Explore Transcript and Drug Patterns in the NCI-60 Cell Line Set
Cancer Res 72:3499-3511. URL:
http://discover.nci.nih.gov/cellminer/analysis.do).
To determine whether a lactate treatment can change the metabolism of
receptor positive and triple negative breast cancer cell lines by analysis of
lactate dehydrogenase enzyme (LDH) isoenzymes.
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