Metabolomic analysis of HER2-positive breast cancer cells

Malika Sahni*, Resmi

Ravindran ,

Imran

Khan ,

Jason Bush*, Krish

# Krishnan

*Department of Biology, of Chemistry, California State University, Fresno Department of Pathology and Laboratory Medicine, University of California, Davis

#Department

INTRODUCTION
Breast cancer is one of the most common diseases in women around the globe. Among various subtypes of breast cancer, HER2-positive accounts for more than 20% of the diagnosed breast cancer cases. Human epidermal growth factor receptor-2 (Her2/neu/p185HER2) is a transmembrane protein tyrosine kinase receptor, encoded by the protooncogene c-erbB-2 (Her2/neu). There is no known ligand specific to HER2 receptor, however, HER2 is always in an active conformation to serve as a co-receptor for heterodimer formation to facilitate signal transduction and activation of various downstream pathways. HER2-positive breast cancer is characterized by aggressive growth and poor prognosis. This is generally due to either the amplification of HER2 gene and/or overexpression of the HER2 protein. However, the biological mechanism(s) are not well understood. Recently, altered metabolism has been identified as an important hallmark of cancer. With the advances in techniques like Nuclear Magnetic Resonance (NMR) spectroscopy, comparative cancer metabolomics has emerged as an important aspect to provide insight into cancer and carcinogenesis.

RESULTS
ERBB2 Gene Expression (NCBI)
250 150 100 75

-HER2

-GAPDH

Figure 2. Expression of ERBB2/HER2 in human breast cancer cell lines. (Left) Gene expression comparison among four cell lines using GEO2R platform (accession GSE12790) (Right) Protein expression levels in H00, H01, H02, H03 cells used for metabolomic experiments and analyses. SKBR3 and MCF7 breast cancer cells were used as positive and negative controls, respectively.

Figure 3. Statistical analyses of NMR (1D) data of H00-H03 sample set. (Left) Partial least squares-discriminant analysis (PLS-DA) shows good discrimination between samples H00, H01 & H02 cells (Right) Non-Parametric (Kruskal Wallis) Test for significance clearly shows increase in Leucine and Isoleucine concentration.

(A)

(B)

LDHA

DUSP6

PTPN1

DUSP10

Figure 1. HER2 signaling pathways. Source: Arteaga et al, 2011.

RESEARCH GOALS
To examine (1) metabolite profiles of HER2-positive breast cancer cell lines and their correlation with their gene and protein expression profiles (2) effect of serum starvation and pervanadate treatment on the metabolic profiles of these HER2-expressing cell lines.

Figure 4. Statistical analyses of NMR (1D) data of S-SS-SSP sample set. (A) Partial Least Squares-Discriminant Analysis (PLS-DA) S, SS & SSP cells (B) NonParametric (Kruskal Wallis) Test for significance shows strong decrease in Lactate concentration in the order S>SS>SSP (C) Corresponding NMR spectrum indicating similar results.

Figure 5. Gene expression profiles. Gene expression comparison among the four experimental cell lines, H03, H02, H01 & H00 using GEO2R platform (NCBI) for lactose dehydrogenase A (LDHA), dual specificity phosphatase 6 (DUSP6)/MKP-3, protein tyrosine phosphatase, non receptor type 1 (PTPN1)/PTP1B and dual specificity phosphatase 10 (DUSP10)/MKP-5.

EXPERIMENTAL APPROACH

DISCUSSION & FUTURE DIRECTIONS

Increased trend in metabolomic expression in the order H03>H02>H01>H00. Metabolomic data correlated with protein and gene expression data.
POTENTIAL BIOMARKERS Lactate PTPN1 LDHA DUSP6 DUSP10 TYPE Metabolite Protein/Phosphatase Protein/Enzyme Protein/Phosphatase Protein/Phosphatase

Leucine and Isoleucine concentration strongly correlates with HER2 protein and gene expression levels. Further studies to validate the data is required.
Serum starvation (SS) and Serum starvation+Pervanadate treatment (SSP) shows a sharp decline in lactate concentration in the order S> SS> SSP.
Gene Expression Metabolomics Protein Expression

Pervanadate is a broad range potent tyrosine phosphatase inhibitor which indirectly increases HER2 tyrosine kinase phosphorylation. High levels of LDHA, PTPN1, DUSP6 & DUSP10 gene expression. These are novel results indicating an alternate role of protein tyrosine phosphatases (PTPs and DUSPs).

Figure 6. Potential biomarkers elucidated in this study. (Bottom) A hypothetical pathway where PTPs/DUSPs positively regulate HER2/ERBB2 signaling and stimulate expression of LDHA leading to high lactate.

PTPs/DUSPs

ERBB2 LDHA LACTATE

NMR sample preparation 1H-1D NMR GEO dataset Analyses Statistical analyses Identification of metabolites Metabolite confirmation -2D TOCSY NMR Pathway validation Western blotting

An intensive pathway analysis is underway to shed more light into HER2 signaling pathways and the role of PTPs and DUSPs in HER2 signaling.

ACKNOWLEDGEMENT
This study has been possible with the collaboration of Dr. Imran Khan at UC Davis. This research is partly funded by NIH (5P20CA138025-03) to JAB and VVK.