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Microarray analysis reveals potential mechanisms of BRMS1-mediated metastasis suppression

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Abstract

We used Affymetrix microarrays to compare gene expression profiles of the metastatic parental breast cancer cell line MDA-MB-435 (435) and the non-metastatic daughter cell line created by the stable expression of the BReast cancer Metastasis Suppressor 1 (BRMS1) gene in 435 cells, MDA-MB-435-BRMS1 (435/BRMS1). Analysis of microarray data provided insight into some of the potential mechanisms by which BRMS1 inhibits tumor formation at secondary sites. Furthermore, due to the importance of the microenvironment, we also examined gene expression under different growth conditions (i.e., plus or minus serum). Expression of 565 genes was significantly (adjusted P-value <0.05) altered regardless of in vitro growth conditions. BRMS1 expression significantly increased multiple major histocompatability complex (MHC) genes and significantly decreased expression of several genes associated with protein localization and secretion. The pattern of gene expression associated with BRMS1 expression suggests that metastasis suppression may be mediated by enhanced immune recognition, altered transport, and/or secretion of metastasis-associated proteins.

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Abbreviations

435:

MDA-MB-435 cells

435/BRMS1:

MDA-MB-435/BRMS1 cells

ABI:

Abl-interactor protein

ANOVA:

Analysis of variance

BP:

Biological process

BRCA:

Breast cancer gene

BRMS:

Breast cancer metastasis suppressor

CASP1:

Caspase 1

CC:

Cellular component

DAG:

Directed acyclic graph

DIAG:

Diacylglycerol

DMEM:

Dulbecco’s modified eagle’s medium

Ece:

Endothelin converting enzyme

EDTA:

Ethylene diamine tetraacetic acid

ENPP:

Ectonucleotide pyrophosphatase phosphodiesterase

ER:

Endoplasmic reticulum

FBS:

Fetal bovine serum

FDR:

False discovery rate

GO:

Gene ontology

Her:

Human epidermal growth factor receptor

HPRT:

Hypoxanthine guanine ribosyltransferase

MHC:

Major histocompatibility complex

NK:

Natural killer cell

NF-κ-B:

Nuclear factor-kappa in B-cells

PBS:

Phosphate buffered saline

PDGF:

Platelet derived growth factor

PKC:

Protein kinase C

pRb:

Retinoblastoma protein

qRT-PCR:

Quantitative real-time polymerase chain reaction

RMA:

Robust multi-array analysis

RNA:

Ribonucleic acid

Th:

Helper T cells

USU:

Utah State University

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Acknowledgements

The authors wish to acknowledge the following organizations for funding: USDA/CSREES Special Research Grant # WY 2004/2005-06084, National Institutes of Health grant # CA87728, and the National Foundation for Cancer Research. We also wish to thank Ning Lin Yin, Ryan Black and Katie Grover of the USU Affymetrix Core Laboratory. We thank Graham Casey (Cleveland Clinic Foundation) and Kent Hunter (N.I.H.) for input. This is Utah Agricultural Experiment Station paper 7864.

Author information

Author notes

  1. Jacob Michaelson

    Present address: Max Planck Institute, Otto-Dix-Ring 72, 01219, Dresden, Germany

Authors and Affiliations

  1. Center for Integrated BioSystems, Utah State University, Logan, UT, 84322-4700, USA

    Patricia J. Champine & Jacob Michaelson

  2. Department of Nutrition and Food Sciences and Center for Integrated BioSystems, Utah State University, Logan, UT, 84322-4700, USA

    Bart C. Weimer

  3. Department of Pathology, Comprehensive Cancer Center, University of Alabama at Birmingham, Birmingham, AL, 35294-0019, USA

    Danny R. Welch

  4. Department of Biology and Center for Integrated BioSystems, Utah State University, Logan, UT, 84322-5305, USA

    Daryll B. DeWald

  5. National Foundation for Cancer Research, Center for Metastasis Research, Bethesda, MD, 20814, USA

    Danny R. Welch & Daryll B. DeWald

Authors
  1. Patricia J. Champine
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Corresponding author

Correspondence to Daryll B. DeWald.

Electronic supplementary material

Appendix

Appendix

Table 1 Genes involved in immune response induced in 435/BRMS cells relative to 435 cells
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Table 2 Genes involved in protein localization/secretion altered in 435/BRMS cells relative to 435 cells
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Table 3 Genes involved in Golgi apparatus altered in 435/BRMS cells relative to 435 cells
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Fig. A.1
figure 4

A flow chart of the analytical approach

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Fig. A.2
figure 5

Heat map of significant genes within the biological process (BP) Gene Ontology (GO) term immune response. Color key is shown at top left and represents the log(2) expression intensity for the average of the probe sets for the specified gene determined to be significantly (adj. P-value <0.05) altered in our data. Cyan color indicates lower relative expression and magenta indicates higher relative expression. If more than one probe set for a given gene was determined to be significant, they were averaged together prior to heat mapping and the number of probe sets is indicated in parentheses to the right of the gene symbol on the map. The presence or absence of fetal bovine serum (FBS) in the samples from each cell line is indicated at the bottom of the map

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Fig. A.3
figure 6

Heat map of significant genes within the biological process (BP) Gene Ontology (GO) term protein localization. Color key is shown at top left and represents the log(2) expression intensity for the average of the probe sets for the specified gene determined to be significantly altered in our data. Cyan color indicates lower relative expression and magenta indicates higher relative expression. If more than one probe set for a given gene was determined to be significant, they were averaged together prior to heat mapping and the number of probe sets is indicated in parentheses to the right of the gene symbol on the map. The presence or absence of fetal bovine serum (FBS) in the samples from each cell line is indicated at the bottom of the map

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Fig. A.4
figure 7

Heat map of significant genes within the cellular component (CC) Gene Ontology (GO) term Golgi apparatus. Color key is shown at top left and represents the log(2) expression intensity for the average of the probe sets for the specified gene determined to be significantly altered in our data. Cyan color indicates lower relative expression and magenta indicates higher relative expression. If more than one probe set for a given gene was determined to be significant, they were averaged together prior to heat mapping and the number of probe sets is indicated in parentheses to the right of the gene symbol on the map. The presence or absence of fetal bovine serum (FBS) in the samples from each cell line is indicated at the bottom of the map

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Fig. A.5
figure 8

Quantitive real-time expression analyses confirm microarray data. Panel A: Quantitative real-time polymerase chain reaction analysis of select gene expression in nanograms of RNA from 435 and 435/BRMS1 cells grown in serum containing media. Standard deviation of each of the three replicates is shown. Panel B: Quantitative real-time polymerase chain reaction analysis of select gene expression with standard deviation in nanograms of RNA from 435 and 435/BRMS1 cells grown in serum-free media. Standard deviation of each of the three replicates is shown. Gene expression was determined relative to standards from both cell lines using the hypoxanthine guanine phosphoribosyl transferase gene

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Champine, P.J., Michaelson, J., Weimer, B.C. et al. Microarray analysis reveals potential mechanisms of BRMS1-mediated metastasis suppression. Clin Exp Metastasis 24, 551–565 (2007). https://doi.org/10.1007/s10585-007-9092-8

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  • DOI: https://doi.org/10.1007/s10585-007-9092-8

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