Success of the human genome project has led to an increased understanding of cancer at the molecular level (Lander et al., 2001
; Venter et al., 2001
). Elucidation of the human genome identified approximately 23,000 genes that encode for 100,000 to 150,000 different transcripts (transcriptome). The functional products, the human proteome, are much more complex with a 10-fold increase in number. Traditional antibody based and target directed analysis is limited to known proteins and is not able to detect, compare and identify hundreds of unknown proteins simultaneously. Proteomics techniques, such as mass spectrometry (MS) coupled with powerful bioinformatic tools, now allow high through-put discovery of novel proteins, and are evolving rapidly to meet the formidable challenge of protein diversity in biomarker research.
Complexity of cancer proteome far exceeds the dynamic range of any single analytical method or instrument, and precludes the identification of most low abundance proteins. In this study, we focused on the hydrophobic sub-proteome to enrich these key low abundance proteins for enhanced biomarker detection. The rationale for choosing hydrophobicity of proteins to enrich cancer biomarkers is based on recent observations reported by Whitelegge et al. (2004)
that many integral membrane proteins elute with low efficiency from polymeric reverse-phase columns (PLRP/S) but hydrophobic proteins retained on the column can be recovered by a formic acid for elution.
In this study, we have demonstrated that hydrophobic fractionation enhances detection of novel membrane proteins by mass spectrometry compared to conventional membrane preparations or whole cell lysate. Hydrophobic columns do not bind highly abundant cytosolic or soluble proteins, such as pyruvate kinase, structural components tubulin and actinin, or serum albumin present in tissue specimens, which allowed us to detect the less abundant proteins of interest. Several proteins usually considered to be non-hydrophobic were found to the hydrophobic matrix. This may be due to: 1). Protein-protein complex formation between a lipophilic membrane protein and cytosolic protein bound tightly that elute with the hydrophobic proteins; 2). Fatty acylation of these proteins or denatured protein exposing a hydrophobic core.
In this study, we have identified a rich source of hydrophobic proteins from selected human tumors and cell lines. Many of these proteins have known important cellular functions, including heat shock proteins (Soo et al., 2008
), translation elongation factors (White-Gilbertson et al., 2008
), EGFR (Charpidou et al., 2008
), cytokeratin (Diaz et al., 2007
), CD44 (Ginestier et al., 2007
), cadherin (Wang et al., 2008
), mitochondrial aldehyde dehydrogenase (Croker et al., 2008
), endothelial cell growth factors (Mohammed et al., 2007
; Relf et al., 1997
), mucin (Rubinstein et al., 2009
), and annexin (Imai et al., 2008
When breast tumors, adjacent normal tissues and cell lines of TNBC and non-TNBC origins were compared, protein expression of the two groups were assigned into 3 categories: upregulated, unchanged, and down-regulated. Since up-regulated proteins are probably more useful as cancer biomarkers and drug targets, we focused our report on these proteins.
The unregulated proteins in TNBC were classified into 7 categories according to their cellular activities: 1). Metabolism related proteins, such as ATP synthase, glutathione transferase, mitochondrial aldehyde dehydrogenase, pyruvate kinase, glucosidase and fatty acid synthase. 2) Growth factors, such as endothelial cell growth factor, which plays an important role in angiogenesis. 3). Protein degradation pathways, such as proteosome subunits, ubiquitin conjugation factors, and ubiquitin-activating enzymes. 4) Transcription and translation regulatory proteins, such as DNA helicase, calreticulin, enolase, eukaryotic translation elongation factors, nucleolin, polymerase I, and transcript release factor, ribosome-binding proteins, DNA topoisomerase, and RNA polymerase. 5) Membrane channel or channel related proteins, such as annexin, voltage-dependent P/Q-type calcium channel subunits. 6) Cell-cell adhesion, which is important in the micro-environment of cancer cells, potentially helps cell migration and cancer metastasis, such as cadherin and CD44. 7) Cellular stress response, heat shock proteins, keratin, and tumor rejection antigen. TNBC specific up-regulation in the 7 functionalities was seen both in cell lines and human cancers, which may help account for the aggressiveness of TNBC. These invasive cancer cells are likely equipped with mechanisms capable of responding to cellular stress, such as hypoxia and nutritional depletion caused by their propensity to out-grow the existing vascular supply.
Annexin related proteins also were highly over-expressed in TNBC, especially in the cell lines with a greater than 100-fold increase. Our findings are in accord with previous reports of annexin over-expression correlating with the aggressiveness of cancer. Annexin A3 has been found to be significantly up-regulated in invasive lung adenocarcinomas with lymph node metastasis compared to those without lymph node metastasis (Liu et al., 2009
). Similarly, annexin is significantly elevated in lymphatic metastasis of mouse hepatocarcinoma (Liu et al., 2008
). Our previous study also showed that altered expression of annexin A1 is correlated with breast cancer development and progression (Shen et al., 2005
; Shen et al., 2006
). Together, these findings provide strong evidence that Annexin family proteins are likely to contribute to the aggressive phenotype and metastatic potential of cancer cells.
Our study also identified over expression of several important stem-cell markers in TNBC cancer specimens and TNBC cell lines compared with non-TNBC samples. For example, CD44 (gi|2507241), a stem cell marker, was found oeverexpressed in both MDAMB231 cells and MDAMB468 cells. CD44, is from a family of transmembrane p-glycoproteins, which are adhesion molecules binding to the extracellular matrix containing hyaluronic acid, collagen, fibronectin, laminin, and FGF-2 (Günthert, 1993
). CD44 has been shown to contribute to both the metastatic potential in pancreatic cancer (Wielenga et al., 1993
; Günthert et al., 1991
) and drug resistance (Li et al., 2008
). Other stem cell markers, such as integrin α6 (gi|12644170, also known as CD49F), and integrin beta-1 precursor (gi|124963, also called CD29), were also found to be over-expressed in MDAMB-231 cells. CD49F is highly-expressed by the basal layer of proliferating skin epithelial cells and by breast cancer stem cells. It regulates cell adhesion to the extracellular matrix and is involved in cancer cell migration, invasion, pathologic angiogenesis and tumor cell survival (Mercurio et al., 2001
; Nikolopoulous et al., 2004
). Another stem-cell marker, aldehyde dehydrogenase (ALDH, gi|62511242) was found in Case A—a TNBC tumor. ALDH1 is a detoxifying enzyme responsible for the oxidation of intracellular aldehydes and may play a role in early differentiation of stem cells through oxidizing retinol to retinoic acid. High levels of ALDH1 activity also have been found in other human stem cells of hematopoietic and neural origin. Because breast cancer stem cells have been implicated in radiation and chemotherapy resistance, as well as increasing the potential of metastasis, these findings may explain treatment failure as well as metastasis that are frequently seen in TNBC patients. The development of an effective therapeutic strategy for this disease may depend on finding a new way to target the stem cell population.
Although still preliminary, cancer proteomic discoveries have shown real promise in improving the understanding of tumor biology. Our study provides evidence that it is possible to identify hundreds of relevant proteins in a selected sub-proteome using only mg of cancer tissue. However, detection of very low abundance proteins remains to be a challenge. Further improvements in protein separation methods coupled with mass spectrometry to isolate different types of proteins and proteins with post-translational modifications may allow deeper profiling of the low abundance proteins in the near future.
Our study demonstrates that hydrophobic fractionation is an effective method to enrich an important class of tumor biomarkers and provides new evidence that LC/MS/MS can identify and quantify differences in cancer-related protein expression. When sufficiently refined, these powerful new technologies may pave the way for earlier detection and better treatment of breast cancer.