G-protein coupled receptor-associated sorting protein 1 (GASP-1), a potential biomarker in breast cancer

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Abstract

An innovative “2-D high performance liquid electrophoresis” (2-D HPLE) technology was used to identify serum biomarkers associated with the early stage of breast cancer in addition to other more advanced stages. 2-D HPLE is a newly developed electrophoretic technology that separates 100s of serum albumin complexes on a polyvinyl membrane based on their surface charges. Association of cancer proteins or their fragments (biomarkers) with pre-existing albumin complexes in the blood of cancer patients results in altered mobility on the membrane. Using 2-D HPLE we identified that a specific fragment of G-protein coupled receptor-associated sorting protein 1 (GASP-1) was present in the sera of patients with early stage disease but absent in sera of normal patients. GASP-1 has been shown to modulate lysosomal sorting and functional down-regulation of a variety of G-protein coupled receptors in neuronal cells. However, no reports have linked GASP-1 to breast cancer pathogenesis. GASP-1 was detected in tumor extracts of 7 cases of Stage 2 and Stage 3 breast cancers, but not in adjacent normal tissue as revealed by western blot analysis using an antibody developed against a GASP-1 peptide identified by our 2-D HPLE technology. Using this antibody, we immunohistochemically detected over-expression of GASP-1 in all of 107 cases of archived ductal breast carcinoma tumor samples, while normal adjacent breast tissue from 12 cases of ductal carcinoma showed little or no staining. Additionally, all 10 cases of metastatic breast carcinoma present in lymph nodes were positive. Strong positive GASP-1 staining was observed in all tumor tissue including ductal carcinoma in situ (DCIS) and invasive ductal carcinoma. Additionally, we observed a wide spectrum of enhanced staining of premalignant ductal epithelial cells present in benign ducts and those found in atypical ductal hyperplasia (ADH). These studies identify GASP-1 as a potential new serum and tumor biomarker for breast cancer and suggest that GASP-1 may be a novel target for the development of breast cancer therapeutics.

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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