G-protein coupled receptor-associated sorting protein 1 (GASP-1), a potential biomarker in breast cancer
Highlights
► A novel method for identifying cancer biomarker-containing serum albumin complexes was developed. ► As breast cancer progresses, more cancer biomarker-containing albumin complexes are formed. ► A cancer peptide motif from GASP-1 was found to be sequestered by serum albumin. ► Over-expression of GASP-1 begins at a precancerous stage and is more pronounced in all later stages.
Introduction
Breast cancer is the second most common cause of cancer death in women in the United States and is also a cause of disability, psychological trauma, and economic loss. Breast cancer morbidity increases significantly if it is not detected early in its progression. Early detection of breast cancer before symptoms appear is the most effective deterrent of breast cancer. It is estimated that between 15 and 25% of women with early stage breast cancer are currently missed by widely used diagnostic procedures such as mammography. The real challenge is to address the inherent limitations of breast cancer detection by identifying new breast cancer markers that can be imaged and detected in the blood. To this end, we have developed a novel approach to detect new breast cancer biomarkers that may be predictive of early stage disease.
The approach is an electrophoretic separation technology that can rapidly detect tumor markers in patient sera. The separation technology, which we refer to as two dimensional high performance liquid electrophoresis (2-D HPLE), can separate tumor protein albumin complexes on a PVDF membrane. This technology is able to isolate biomarkers from body fluids (serum, cerebrospinal fluids, etc.) under non-denaturing conditions. When mass spectrometric analysis (LC/MS/MS) is coupled with albumin complexes separated by 2-D HPLE, the dynamic range of detection is enhanced to the 1010 range required for detecting low copy number cancer biomarkers. The separation of albumin complexes by 2-D HPLE is based on their net charge or isoelectric points (pI). Association of a newly produced cancer peptide fragment with a pre-existing albumin complex changes its pI and this new complex will migrate to a different location on the PVDF membrane, allowing its detection among hundreds of already present albumin complexes. Using the 2-D HPLE technology, we have identified over 50 novel biomarkers in breast, pancreatic, liver and skin cancer (unpublished data).
In this paper we show that one of these biomarkers that is expressed in the blood of early stage breast cancer patients but absent in the sera of normal healthy individuals is highly expressed in breast tumors. The biomarker that we have identified is G-protein coupled receptor-associated sorting protein 1 (GASP-1). GASP-1 modulates lysosomal sorting, receptor recycling and functional down-regulation of a variety of G-protein coupled receptors. It is involved in silencing signals by targeting receptors for degradation in lysosomes or amplifying signals by promoting endosome formation (Whistler et al., 2002). Previous studies have shown that GASP-1 interacts with cytoplasmic tails of a variety of G-protein coupled receptors such as D2 dopamine receptor/DRD2, delta opioid receptor/OPRD1, beta-2 adrenergic receptor/ADRB2 and D4 dopamine receptor/DRD4 (Simonin et al., 2004). However, no studies have linked GASP-1 to breast cancer pathogenesis.
Section snippets
Separation of serum protein complexes from breast cancer patients using 2-D HPLE
We have developed an innovative procedure for separation and detection of protein complexes containing the newly produced breast cancer markers. To retain protein complexes containing low-abundance breast cancer markers, the electrophoresis process was carried out under non-denaturing conditions. A two-dimensional high performance liquid electrophoresis (2-D HPLE) technology was developed for this purpose (Chang and Yonan, 2008). Unlike the commonly used 2-D PAGE (O'Farrell, 1975), proteins and
Identification of serum albumin complexes associated with early and later stages of breast cancer by 2-D HPLE
Serum samples from patients with different stages of breast cancer as well as normal controls were purchased from the Lombardi Comprehensive Cancer Center at Georgetown University. They house an extensive serum and tumor repository of breast cancer patients. As described in Material and methods, 2-D HPLE separates serum albumin complexes and can detect any newly formed cancer-protein containing albumin complexes among several hundred pre-existing complexes due to change in their surface charges
Discussion
G-protein-coupled receptors (GPCRs) represent one of the most abundant receptor networks encoded by the human genome and these receptors are targeted for the treatment of various diseases by more than 40% of the drugs sold by the pharmaceutical industry (Abu-Helo and Simonin, 2010). These GPCRs are tightly regulated by a vast repertoire of GPCR interacting proteins. These interacting proteins have important functions, including the targeting of receptors to specific subcellular compartments,
Acknowledgments
This study was supported in part by a grant from the Pennsylvania State Commonwealth Universal Research Enhancement (CURE) Program.
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