Protein-based markers that classify tumor subtypes and predict therapeutic response would be clinically useful in guiding patient treatment. We investigated the LC-MS/MS-identified protein biosignatures in 39 baseline breast cancer specimens including 28 HER2-positive and 11 triple-negative (TNBC) tumors. Twenty proteins were found to correctly classify all HER2 positive and 7 of the 11 TNBC tumors. Among them, galectin-3-binding protein and ALDH1A1 were found preferentially elevated in TNBC, whereas CK19, transferrin, transketolase, and thymosin
Chemotherapy has long been used to treat all types of cancer. Although survival benefits from adjuvant systemic chemotherapy in breast cancer have been thoroughly documented [
In selective subtypes of breast cancer, therapies targeting specific signal transduction and/or metabolic pathways have been successful. For example, Herceptin for HER2/neu positive breast cancer [
Breast cancer is a heterogeneous disease molecularly, histologically, and clinically. Clinical outcomes from the same treatment vary widely even among patients with tumors of identical stage and histology. Breast cancers developed from an accumulation of genetic alterations may partially explain the differences observed including tumor responses to anticancer agents [
Proteomics has been employed in recent years to identify new disease-related biomarkers for cancer diagnosis and implementation of tailored treatment [
Previously we used SELDI mass spectrometry to profile tumor response to neoadjuvant treatment and found that significant m/z profile differences existed between cancers of nonresponders (tumor regression rate ≤25%) and others (tumor regression rate >25%) [
Breast tumors were collected, processed, and banked as previously described [
Protein extraction from tumors and depletion of abundant proteins from tumor lysates were performed as previously described [
Because the blood proteins in the breast cancer tissue can cause significant ion suppression of lower abundance cancer-related proteins/peptides which may mask ion signals of less abundant peptides with similar M/Z ratios and retention times. In addition, the over presentation of serum proteins in the specimen may lower the amount of the cancer-related proteins available for LC-MS/MS analysis [
The dried protein samples were dissolved in 6 M guanidine HCl, reduced with DTT (5 mM–15 mM), and alkylated using 10 mM iodoacetamide. Samples were then diluted with NH4HCO3 to lower guanidine HCl concentration (1 M), mixed with trypsin (1 : 50 w/w ratio, sequencing grade, Promega) containing 50 mM ammonium bicarbonate, and incubated at 37°C overnight. Samples were desalted by C18 Microspin columns (The Nest Group), and the eluates were dried in a vacuum centrifuge.
Each digested and dried sample was prepared for LC-MS/MS analysis as previously reported [
BioWorks software (version 3.3.1, Thermo Fisher Sceintific), based on the SEQUEST algorithm (SRF v.5, Thermo Fisher Scientific), was used to search the mass spectra against a human trypsin indexed database (human.fasta.hdr database, Version 12.2, 227246 entries) as described by Whelan et al. [
From the resulting MS/MS protein identifications, a list of proteins was generated for each sample. A list of semi-quantitative protein abundances in the different samples was developed using the normalized spectrum counts of the identified tryptic peptides from each protein, as compiled by the Scaffold program. The protein lists and their relative abundances were then compared to find differentially expressed proteins between two groups.
The data files exported from Scaffold were further processed as Excel files. The top 60% (180) abundant proteins of the 315 identified proteins were further selected for hierarchical clustering and supervised classification studies. Those proteins with at least a 2-fold difference in mean spectral counts between any two groups were selected for analysis in the web-based Gene Expression Profile Analysis Suite (GEPAS, version 4.0,
A small portion of each of the baseline tumors was embedded in OCT and stored at −80°C. Endogenous peroxidase activity was quenched with 0.6% hydrogen peroxide in methanol for 10 minutes, and endogenous biotin was eliminated by Biotin Blocking System (DAKO, x0590). After blocking with 1 : 5 diluted normal goat serum or fetal bovine serum, slides were incubated for 1 hour with primary antibody (CK19, mouse IgG, ready to use, DAKO; galectin-3-binding protein, Goat IgG, 1 : 200 dilution, R&D) and 30 minutes with biotinylated secondary antibody (biotinylated anti-mouse Ig, 1 : 800 dilution, DAKO; biotinylated anti-goat Ig, 1 : 200 dilution, Vector Labs). Antigen-antibody complexes were then detected by the StreptABComplex/HRP method (DAKO) using diaminobenzidine as a chromogenic substrate (DAKO). Immunostained slides were lightly counterstained with hematoxylin. For negative controls, primary antibodies were replaced by mouse IgG or goat IgG.
The reported thirty-nine baseline tumor specimens included 28 HER2-positive breast cancers and 11 TNBC with HER2 status determined by fluorescence in situ hybridization (FISH) assay. Fifteen of the 28 HER2+ patients were randomized to receive TC, and the remaining 13 received TC and Herceptin (TCH) before surgery. All eleven patients with TNBC received neoadjuvant TC. Following the neoadjuvant treatment, 28 patients with HER2+ tumors showed 12 responders (R), including 7 with pathological complete response (pCR) and 5 with a tumor regression rate >75%, 12 intermediate responders (IR), and 4 nonresponders (NR). In the TNBC group, there were 7 responders including 6 pCR and 1 with tumor regression rate >75%, 3 IR, and 1 NR. The clinical characteristics and pathologic features of the 11 TNBC and 28 HER2+ cases are summarized in Tables
Clinical characteristics of 11 TNBC tumors
LTQ Orbitrap sample ID | Patient age | Ethnicity | TR % | Response | T stage | Histological type | ER | PR | FISH R/G ratio | Neoadjuvant |
---|---|---|---|---|---|---|---|---|---|---|
#1 | 61 | White | 80 | R | T3 | IDC | − | 1.10 | TC | |
#2 | 29 | Hispanic | −60 | NR | T3 | IDC | − | − | 0.92 | TC |
#5 | 55 | Hispanic | 100 | R (pCR) | T3 | IDC | − | − | 0.92 | TC |
#6 | 54 | Hispanic | 100 | R (pCR) | T3 | IDC | − | − | 1.01 | TC |
#7 | 40 | Asian | 45 | IR | T3 | IDC | − | − | 1.17 | TC |
#8 | 44 | White | R (pCR) | T3 | IDC | − | − | 1.00 | TC | |
#9 | 49 | Hispanic | 48 | IR | T4 | IDC | − | − | 1.10 | TC |
#10 | 53 | White | 100 | R (pCR) | T3 | IDC | − | − | 1.20 | TC |
#11 | 84 | Asian | 100 | R (pCR) | T4 | IDC | − | − | 1.03 | TC |
#36 | 45 | Hispanic | 30 | IR | T3 | IDC | − | − | 1.27 | TC |
#37 | 38 | White | 100 | R (pCR) | T2 | IDC | − | − | 1.10 | TC |
aLN positive without residual primary cancer.
Clinical characteristics of 28 HER2+ tumors
LTQ Orbitrap sample ID | Patient age | Ethnicity | TRR % | Response | T stage | Histological type | ER | PR | FISH R/G ratio | Neoadjuvant |
---|---|---|---|---|---|---|---|---|---|---|
#17 | 38 | White | 40 | IR | T3 | IDC | + | − | 12.4 | TC |
#18 | 63 | Asian | 100 | R (pCR) | T3 | IDC | + | − | 12.7 | TCH |
#19 | 57 | White | 100 | R (pCR) | T3 | IDC | − | − | 4.6 | TC |
#20 | 56 | Asian | 78.2 | R | T4 | IDC | + | − | 10.71 | TC |
#21 | 51 | Black | 56 | IR | T3 | IDC | − | − | 19.97 | TCH |
#22 | 31 | White | 45.5 | IR | T3 | IDC | + | + | 2.2 | TC |
#23 | 55 | White | 80 | R | T4 | IDC | + | + | 3.8 | TC |
#24 | 45 | Asian | 75 | IR | T4 | IDC | + | + | 2.7 | TCH |
#25 | 42 | Hispanic | 63.5 | IR | T4 | IDC | + | − | 2.5 | TC |
#26 | 50 | White | 67.1 | IR | T3 | IDC | − | − | 2.41 | TCH |
#27 | 33 | White | 82.9 | R | T3 | IDC | + | + | 3.03 | TCH |
#28 | 40 | White | 66.7 | IR | T3 | IDC | − | − | 8.1 | TC |
#29 | 35 | Hispanic | R (pCR) | T3 | IDC | − | − | 42.2 | TCH | |
#30 | 44 | White | 97.3 | R | T4 | IDC | − | − | 4.2 | TC |
#31 | 30 | White | −7.7 | NR | T3 | IDC | + | − | 5 | TC |
#32 | 57 | White | 25% | NR | T4 | IDC | + | − | >4 | TCH |
#33 | 37 | White | 33.3 | IR | T2 | IDC | + | + | 9.49 | TC |
#34 | 36 | Black | 25 | NR | T3 | IDC | − | − | 5.1 | TC |
#35 | 42 | White | 60 | IR | T2 | IDC | + | + | 4.5 | TCH |
#38 | 55 | White | 42.3 | IR | T4 | IDC | − | − | 3.9 | TCH |
#39 | 47 | White | R (pCR) | T3 | IDC | + | + | >20 | TCH | |
#40 | 50 | Asian | R (pCR) | T3 | IDC | + | + | 4.19 | TCH | |
#41 | 58 | White | 50 | IR | T4 | IDC | + | + | 2.1 | TCH |
#42 | 40 | Asian | 60 | IR | T2 | IDC | − | − | 16 | TC |
#43 | 37 | White | −85.6 | NR | T3 | IDC | + | + | 3.1 | TC |
#44 | 49 | White | R (pCR) | T2 | IDC | + | − | 7.7 | TCH | |
#45 | 55 | Asian | 92.6 | R | T3 | IDC | − | − | 9.9 | TC |
#46 | 41 | Hispanic | 100 | R (pCR) | T2 | IDC | − | − | 9.2 | TC |
aLN positive and residual DCIS; bresidual DCIS only.
Proteins identified by MS/MS from the 39 tumors showed that 48 proteins were only found in HER2+ tumors, 24 were only seen in TNBC, and 243 proteins were shared by both, but the quantity of the shared proteins differed widely in the two tumor types. In this study, we focused the analysis on the top 60% abundant proteins (180/315) detected in the 39 tumors.
The 20 most abundant shared proteins by both subtypes of cancer were summarized in Table
The 20 most abundant proteins shared by both HER2-positive and TNBC tumors.
Identified proteins | Accession no. | MW |
---|---|---|
Apolipoprotein A-I | gi∣90108664 | 28 kDa |
Vimentin | gi∣62414289 | 54 kDa |
Enolase 1 | gi∣4503571 | 47 kDa |
Alpha-1 antitrypsin | gi∣157086955 | 45 kDa |
Triosephosphate isomerase 1 | gi∣4507645 (+2) | 27 kDa |
Cyclophilin A | gi∣1633054 | 18 kDa |
Apolipoprotein D | gi∣619383 | 28 kDa |
Cofilin 1 | gi∣5031635 | 19 kDa |
Chaperonin | gi∣31542947 | 61 kDa |
Transgelin 2 | gi∣4507357 | 22 kDa |
Heat shock 70 kDa protein 5 | gi∣16507237 | 72 kDa |
Tumor rejection antigen (gp96) 1 | gi∣4507677 | 92 kDa |
S100 calcium-binding protein A11 | gi∣5032057 | 12 kDa |
Lumican precursor | gi∣4505047 | 38 kDa |
Tropomyosin 4 | gi∣4507651 | 29 kDa |
ATP synthase, H+ transporting, mitochondrial F1 complex | gi∣32189394 | 57 kDa |
Prosaposin isoform a preproprotein | gi∣11386147 | 58 kDa |
Profilin | gi∣157838211 (+4) | 15 kDa |
Heat shock 70 kDa protein 8 isoform 1 | gi∣5729877 | 71 kDa |
Annexin 5 | gi∣4502107 | 36 kDa |
Of the 180 top abundant proteins observed in the 39 breast cancer specimens, 61 were found to have a ≥2-fold difference of spectrum counts between the two subtypes of breast cancer (HER2+ versus TNBC). Because some of these proteins were not detected in every sample, we further refined the list of differential proteins by selecting only those detected in ≥50% of the cases in either group. The selected 44 differentially expressed proteins were tested by hierarchical clustering to classify HER2+ breast cancer versus TNBC. These differentially expressed proteins correctly classified all 28 HER2+ tumors and 8 of the 11 TNBC by unweighted pair-group method using arithmetic average (UPGMA) (Figure
Heat map displaying the expression of 44 proteins in 28 HER2-positive and 11 TNBC tumors. Classification of 39 breast cancer cases into 2 groups based on tumor subtypes (HER-positive tumors and TNBC tumors) by the hierarchical clustering using GEPAS software. Each column represents a case as labeled on top, the short labeling cases are “TNBC” with sample ID, and long labeling cases are “HER2-positive” with sample ID. Each row represents a protein ID as indicated at the right. 44 proteins were expressed by ≥2-fold differences and detected in ≥50% of the cases in either group.
Self-validation of selected proteins in tumor classification was tested using a supervised classification. The 44 differentially expressed proteins were used to build a model separating subtypes of the tumors by Prophet, a web interface from the Gene Expression Profile Analysis Suite. Error rates were calculated as the number of misclassified tumors divided by total tumor cases tested. The error rates using various numbers of proteins by different models (SVM, KNN, DLDA, PAM, and SOM) were estimated by leaving-one-out tests (see File A in Supplementary Material available online at doi:10.1155/2011/896476). SVM had the lowest error rate (10%, 4/39) with 90% accuracy in tumor classification. The top 20 protein candidates (Table
Top 20 differentially expressed proteins selected by supervised classification methods for classifying two tumor subtypes.
Rank | Accession no. | Protein name | MW | HER2+/TNBC mean | Subcellular location | Function |
---|---|---|---|---|---|---|
1 | gi∣10946578 | Thymosin | 5 kDa | 2.99 | Cytoplasm, cytoskeleton | For cytoskeletal binding, involved in cell growth and maintenance |
2 | gi∣4507521 | Transketolase | 68 kDa | 4.20 | Cytosol | Involved in metabolism. Associated with cell proliferation of uterine and cervical cancer. |
3 | gi∣1633054 | Cyclophilin A | 18 kDa | 2.45 | Cytoplasma | Involved in accelerate the folding of proteins |
4 | gi∣73858568 | Complement component 1 inhibitor | 55 kDa | 0.33 | Secreted | Regulating the complement cascade |
5 | gi∣4557871 | Transferrin | 77 kDa | 16.38 | Secreted | Essential for cell growth and iron-dependent metabolic processes |
6 | gi∣90111766 | Keratin type I cytoskeletal 19 | 44 kDa | 11.29 | Cytoskeleton | Involved in metastatic progression of breast cancer |
7 | gi∣10863895 | Thymosin | 5 kDa | 2.25 | Cytoplasm, cytoskeleton | For cytoskeletal binding, involved in cell growth and maintenance |
8 | gi∣5031863 | Galectin-3-binding protein | 65 kDa | 0.41 | Secreted | Modulating cell-cell and cell-matrix interactions |
9 | gi∣4505753 (+1) | Phosphoglycerate mutase 1 | 29 kDa | 2.51 | Cytosol | Involved in glycolysis |
10 | gi∣5174391 | Aldo-keto reductase family 1, member A1 | 37 kDa | 0.30 | Cytosol | Involved in the reduction of biogenic and xenobiotic aldehydes |
11 | gi∣21361176 | Aldehyde dehydrogenase 1A1 | 55 kDa | 0.39 | Cytoplasm | Detoxifying enzyme responsible for oxidating of intracellular aldehydes. A marker for cancer stem cells |
12 | gi∣4505185 | Macrophage migration inhibitory factor | 12 kDa | 0.36 | Secreted, cytoplasm | Involved in integrin signaling pathways |
13 | gi∣4507645 (+2) | Triosephosphate isomerase 1 | 27 kDa | 2.49 | Cytosol, nucleus | Fatty acid biosynthesis, gluconeogenesis, glycolysis, lipid synthesis |
14 | gi∣4930167 | Aldolase A | 39 kDa | 6.41 | Extracellular, cytoskeleton | Involved in glycolysis |
15 | gi∣116241280 | Adenylyl cyclase-associated protein 1 (CAP 1) | 52 kDa | 3.03 | Membrane | Regulating filament dynamics, cell polarity and signal transduction, |
16 | gi∣21624607 (+5) | Coactosin-like 1 | 16 kDa | 0.42 | Cytoplasm, cytoskeleton | Regulating the actin cytoskeleton |
17 | gi∣160420317 | Filamin A, alpha isoform 2 | 281 kDa | 3.10 | Cytoplasm | Anchoring transmembrane proteins to the actin cytoskeleton, scaffold for cytoplasmic signaling proteins |
18 | gi∣6005942 | Valosin-containing protein | 89 kDa | 3.26 | Cytosol, nucleus | Fragmentation of Golgi stacks during mitosis and reassembly |
19 | gi∣5174539 | Cytosolic malate dehydrogenase | 36 kDa | 2.52 | Cytoplasm | Involved glycolysis, oxidation reduction, and tricarboxylic acid cycle |
20 | gi∣33286418 (+2) | Pyruvate kinase 3 | 58 kDa | 6 | Cytoplasm, nucleus | Involved in glycolysis |
Representative proteins differentially expressed by HER2+ and TNBC tumors. (a)–(c): proteins preferentially expressed in TNBC. (d)-(e): proteins preferentially expressed in HER2+ tumors.
ALDH1A1
Galectin-3-binding protein
Complement component 1 inhibitor
Cytokeratin-19
Transketolase
Transferrin
Of the 28 HER2+ tumors, there were 12 R (including 7 with pCR), 12 IR, and 4 NR. We compared proteomic differences between the two groups with extreme tumor response (pCR and NR) and found that 48 of the 180 proteins had an expressional difference ≥2-fold between 7 pCR versus 4 NR tumors. Self-validation of these potential marker proteins by five supervised classification methods suggested that the KNN had the lowest error rate (9%, 1/11) in predicting tumor response (Files B and C). By using KNN = 1 method, 100% (4/4) NR and 85.7% (6/7) pCR were correctly grouped by 20 selected proteins (Table
Top 20 proteins predicting tumor response to neoadjuvant treatment in HER2-positive tumors.
Rank | Protein name | Accession no. | pCR/NR mean | Subcellular location | Function |
---|---|---|---|---|---|
1 | Enolase 1 | gi∣4503571 | 2.59 | Cytoplasm, cell membrane | Multifunctional enzyme |
2 | Heterogeneous nuclear ribonucleoprotein A2/B1 isoform B1 | gi∣14043072 | 3.51 | Nucleus, cytoplasm | Pre-mRNA processing |
3 | Heat shock 70 kDa protein 1 | gi∣75061728 | 0.24 | Cytoplasm | Stress response |
4 | Vimentin | gi∣62414289 | 9.94 | Cytosol | Class III intermediate filaments |
5 | Vesicle amine transport protein 1 | gi∣18379349 | 0.50 | Cytoplasmic vesicle membrane | Neurotransmitter transport |
6 | Coronin, actin-binding protein, 1A | gi∣5902134 | 2.00 | Cytoplasm | Component of the cytoskeleton of highly motile cells |
7 | Fatty acid-binding protein 4 | gi∣4557579 (+1) | 0.23 | Cytoplasm, nucleus | Lipid transport protein |
8 | Peroxiredoxin 5 | gi∣15826629 | 0.37 | Mitochondrion, cytoplasm, peroxisome | Antioxidant, oxidoreductase peroxidase |
9 | Heat shock 70 kDa protein 9 | gi∣24234688 | 0.15 | Mitochondrion | Control of cell proliferation and cellular aging |
10 | Leucine aminopeptidase 3 | gi∣41393561 | 2.94 | Cell membrane, secreted | Cell-cell signaling |
11 | Apolipoprotein D | gi∣619383 | 2.90 | Secreted | Lipid metabolic process |
12 | L-plastin | gi∣4504965 | 3.14 | Cytoplasm, cell membrane | Activation of T cells, intracellular protein transport |
13 | Anterior gradient protein 2 homolog precursor | gi∣5453541 | 0.11 | Secreted, endoplasmic reticulum | Mucus secretion |
14 | Heat shock 10 kDa protein 1 | gi∣4504523 | 0.37 | Mitochondrion | Stress response |
15 | ATP synthase, H+ transporting, mitochondrial F1 complex | gi∣4757810 | 0.41 | Mitochondrion | Proton-transporting ATP synthase complex assembly |
16 | Glutathione transferase | gi∣20664358 (+5) | 3.29 | Cytoplasm | Glutathione metabolic process |
17 | Chaperonin | gi∣31542947 | 0.33 | Mitochondrion | Stress response |
18 | Complement component 3 precursor | gi∣115298678 | 3.00 | Secreted | Activation of the complement system |
19 | Heterogeneous nuclear ribonucleoprotein D isoform a | gi∣14110420 | 2.19 | Nucleus, cytoplasm | Transcription regulation |
20 | Malate dehydrogenase | gi∣6648067 (+1) | 0.22 | Cytoplasm | Tricarboxylic acid cycle |
Among the 11 TNBC cases, there were 7 R (including 6 pCR), 3 IR, and 1 NR. Due to the small sample size, the proteins of responders’ tumor (R) were compared to all the remaining tumors with less response (IR + NR). Sixty-three of 180 proteins had a ≥2-fold mean differences between the two groups of TNBC with different response to the same treatment. Self-validation of these proteins by five supervised classification methods was used to compare the accuracy in predicting a tumor response. Using DLDA method, 6 of 7 tumors in the R group and 3 of 4 tumors in IR/NR group were correctly classified by the 30 selected proteins (error rate 18%) (Files D and E). Of these 30 proteins, the increased heat shock 70 kDa protein 8, periostin, Ras homolog gene family member A (RhoA), actinin alpha 4, cathepsin D preproprotein, annexin 1, and several other proteins were associated with drug resistance in TNBC (Table
Top 30 proteins predicting tumor response to neoadjuvant chemotherapy in TNBC tumors.
Rank | Protein name | Accession no. | R/IR + NR mean | Subcellular location | Function |
---|---|---|---|---|---|
1 | Heat shock 70 kDa protein 8 isoform 1 | gi∣5729877 | 0.32 | Stress response | |
2 | Periostin precursor (PN) (osteoblast-specific factor 2) | gi∣93138709 | 0.31 | Nucleus | Transcription regulation |
3 | Cyclophilin A | gi∣1633054 | 0.41 | Secreted | Cell attachment adhesion and spreading |
4 | Tyrosine 3/tryptophan 5-monooxygenase activation protein | gi∣5803225 (+1) | 3.71 | Nucleus | Protein binding |
5 | Profilin | gi∣157838211 (+4) | 0.32 | Cytoplasm, cytoskeleton | Actin cytoskeleton organization |
6 | Cardiac muscle alpha actin 1 proprotein | gi∣4885049 | 0.08 | Cytoplasm, cytokeleton | actin filament-based movement, apoptosis |
7 | Beta actin | gi∣4501885 | 0.22 | Cytoplasm, cytokeleton | Cell motility |
8 | Caldesmon (CDM) | gi∣2498204 | 0.42 | Cytoplasm, cytokeleton | Actin- and myosin-binding protein |
9 | Tubulin | gi∣7106439 | 0.19 | Cytosol | Major constituent of microtubules |
10 | Tropomyosin 2 (beta) isoform 1 | gi∣42476296 | 0.11 | Cytoplasm, cytokeleton | Binding to actin filaments |
11 | Actinin, | gi∣12025678 | 0.11 | Nucleus, cytoplasm | Protein transport |
12 | Ras homolog gene family, member A (RhoA) | gi∣10835049 (+4) | 0.33 | Cytoplasm, cell membrane | Regulating a signal transduction pathway |
13 | Heterogeneous nuclear ribonucleoprotein K | gi∣13384620 | 0.33 | Cytoplasm, nucleus | Pre-mRNA-binding proteins |
14 | Tubulin | gi∣6755901 | 0.36 | Cytosol | Major constituent of microtubules |
15 | Tropomyosin 4 | gi∣4507651 | 0.35 | Cytoplasm, cytokeleton | Binds to actin filaments |
16 | Complement component 1 inhibitor precursor | gi∣73858568 | 4.57 | Secreted | Complement pathway |
17 | ATP synthase, H+ transporting, mitochondrial F1 complex | gi∣4757810 | 0.43 | Mitochondrion | Proton-transporting ATP synthase complex assembly |
18 | Calnexin precursor | gi∣10716563 | 0.42 | Endoplasmic reticulum membrane, cell membrane | Calcium-binding protein |
19 | Eukaryotic translation elongation factor 1 alpha 1 | gi∣4503471 | 0.29 | Cytoplasm | Protein biosynthesis |
20 | Annexin I | gi∣4502101 | 0.25 | Nucleus, cytoplasm, membrane | Calcium/phospholipid-binding protein |
21 | Triosephosphate isomerase 1 | gi∣4507645 (+2) | 0.35 | Cytosol, nucleus | Fatty acid biosynthesis, gluconeogenesis, glycolysis, lipid synthesis |
22 | Cathepsin D preproprotein | gi∣4503143 | 0.35 | Lysosome | proteolysis |
23 | Alpha glucosidase II alpha subunit isoform 2 | gi∣38202257 | 0.19 | Cytosol | Glycan metabolism, N-glycan metabolism |
24 | Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein | gi∣4507949 (+1) | 0.42 | Nucleus | Protein binding |
25 | Thymosin | gi∣10863895 | 0.42 | Cytoplasm, cytokeleton | cytoskeleton organization |
26 | Aconitase 2 precursor | gi∣4501867 | 0.44 | Mitochondrion | Carbohydrate metabolism, tricarboxylic acid cycle |
27 | Heterogeneous nuclear ribonucleoprotein D isoform a | gi∣14110420 | 0.48 | Nucleus, cytoplasm | Transcription regulation |
28 | Serine (or cysteine) proteinase inhibitor | gi∣32454741 | 0.25 | Secreted | Inhibits activated protein C, plasminogen activator |
29 | Lumican precursor | gi∣4505047 | 0.28 | Secreted | Binds to laminin |
30 | Apolipoprotein D | gi∣619383 | 2.86 | Secreted | Transport |
CK19 and G3BP protein expressions were tested in breast cancer tumors by immunohistochemistry. The CK19 and G3BP staining showed cytoplasmic/membrane staining pattern in breast cancer cells. The overexpression of CK19 was found in HER2+ breast tumors (Figure
CK19 expressions detected by LC-MS/MS and immunohistochemical (IHC) staining. Elevated CK19 expressions found in HER+ tumor group by LC-MS/MS and confirmed by IHC in most of the frozen HER2+ tumors. (a) Normalized spectrum count of CK19 detected in 39 breast cancer tissues. (b) Immunohistochemical staining of CK19 in a HER2+ frozen tumor (power 200x). (c) Immunohistochemical staining of CK19 in a TNBC frozen tumor (power 200x).
Cytokeratin-19
G3BP expressions detected by LC-MS/MS and immunohistochemical (IHC) staining. Elevated G3BP expressions found in TNBC group by LC-MS/MS and confirmed by IHC in most of the frozen TNBC tumors. (a) Normalized spectrum count of G3BP detected from 39 breast cancer tissues. (b) Immunohistochemical staining of G3BP in a TNBC frozen tumor (power 200x). (c) Immunohistochemical staining of G3BP in a HER2+ frozen tumor (power 200x).
Galectin-3-binding protein
In this discovery study, the MS-detected proteomic differences between two subtypes of breast cancer (HER2+ versus TNBC tumors) were explored, and proteomic prediction of tumor response to neoadjuvant chemotherapy was investigated. LC-MS/MS data sets of proteins from the 39 tumors analyzed allowed us to identify several candidate proteins that could classify tumor subtypes and predict tumor response to neoadjuvant chemotherapy.
Two clinical subtypes of breast cancer, HER2-positive and triple-negative breast cancers, defined by immunohistochemical staining and fluorescence in situ hybridization of three biomarkers of breast cancer have also been confirmed by gene analysis as two distinctive types of breast cancer. In this study, we reported that proteomic analysis could also separate the two subtypes by the unique biosignature associated with each type of breast cancer (Table
Through an extensive literature review, some of the identified proteins have reported roles that are relevant to cancer biology and treatment. In TNBC tumors, we observed that the levels of G3BP, ALDH1A1, and complement component 1 inhibitor protein were preferentially elevated. All of them have been reported to have important biological properties in cancer progression. G3BP, also known as 90
One protein correlated with triple-negative breast cancer meriting a discussion is ALDH1A1, a detoxifying enzyme responsible for oxidizing intracellular aldehydes. This process is important in early differentiation of stem cells through conversion of retinol to retinoic acid [
A different set of proteins was found preferentially elevated in HER2+ tumors. This list included CK19, transferrin, transketolase, and thymosin
Cytokeratins are known to be important in cellular motility, signaling, and division. While CK8/CK18 were similarly detected in both HER2+ and TNBC tumors, elevated CK19 was more commonly found in HER2+ tumors. Our observation coincides with the finding reported by Schulz et al. using a combination of 2D-DIGE/mass spectrometry and western blot [
Transferrin, another protein associated with HER2-positive cancer, is essential for cell growth and iron-dependent metabolic activities including DNA synthesis, electron transport, and mitogenic signaling pathways [
Thymosin
In this study, we also reported the MS-identified protein signature predicting drug-induced tumor response in HER2-positive tumors. We found that enolase 1, vimentin, L-plastin, and ApoD predicted a favorable response of HER2-positive tumors. In contrast, elevated peroxiredoxin 5 and heat shock proteins 70 were found in nonresponding HER2-positive tumors.
Enolase 1(ENO1), a phosphopyruvate dehydratase, is a key glycolytic enzyme involved in anaerobic metabolism under hypoxic conditions of cancer growth, and a cell surface plasminogen receptor for tumor invasion. Overexpression of ENO1 in breast, and lung cancers is associated with tumor progression and rapid tumor growth [
Vimentin is a member of the intermediate filament family. Along with microtubules and actin microfilaments, vimentin is an integral component of the cell cytoskeleton. In cancer, altered vimentin level is associated with a dedifferentiated phenotype, increased motility, invasiveness, and poor clinical prognosis [
In contrast to those molecules associated with favorable tumor response to neoadjuvant therapy, high levels of Prx V in HER2-positive breast cancers were found to be associated with poor response to the same chemotherapy regimen. Peroxiredoxins (Prxs) represent a novel group of peroxidases containing high antioxidant activity involved in cell differentiation and apoptosis [
While some molecules are unique to the characteristics of individual subtype of breast cancer, Hsp70 overexpression was found by us to be associated with drug resistance in both HER2-positive and TNBC tumors. Heat shock proteins are overexpressed in a wide range of human cancers and are implicated in tumor cell proliferation, differentiation, death, invasion, metastasis, and immune recognition [
In TNBC tumors, a list of different proteins was found to be overexpressed in tumors resistant to neoadjuvant chemotherapy. In addition to Hsp70, proteins such as periostin precursor (OSF-2), RhoA, actinin
Periostin was originally identified in a mouse osteoblastic cell line as an extracellular matrix adhesion protein for pre-osteoblasts. In addition to forming bones, teeth, and heart, periostin was recently found to be overexpressed in various types of human cancer. Periostin interacts with multiple cell-surface receptors (most notable integrins) and signals via the PI3-K/Akt and other pathways to promote cancer cell survival, epithelial-mesenchymal transition, invasion, and metastasis [
RhoA is a member of the Ras superfamily. It is involved in the regulation and timing of cell division. It is a small GTPase protein known to regulate the actin cytoskeleton in the formation of stress fibers. RhoA protein levels were significantly increased in breast cancer compared with the matched normal tissue. It has been reported by Fritz et al. that an elevated RhoA protein level correlated with increasing breast tumor grade and poor prognosis [
Actinin
Cathepsin D, an acid protease, is active in intracellular protein breakdown and is involved in the pathogenesis of several diseases. Its preproprotein secreted by cancer cells, acting as a mitogen on both cancer and stromal cells, stimulates both proinvasive and prometastatic properties of cancer cells. Many studies found that cathepsin D preproprotein/cathepsin D level represents an independent prognostic factor in a variety of cancers and is, therefore, considered to be a potential target for anticancer therapy [
Although many proteins identified in this pilot study are interesting with promising potential, this study has several limitations. First, the tumors used in this study were collected from a clinical trial which provided many controlled clinical data; however, the sample size available for proteomic analysis was small. As a result, the findings derived from a small sample size always warrant a cautious interpretation. Second, the HER2-positive group consisted of tumors with different ER and PR status which might interfere with the conclusion. The potential false associations with HER2 might be solved by stratifying the HER2-positive tumors according to hormonal receptor status in a larger study. Lastly, the HER2-positive patients in this study were randomized to receive either chemotherapy alone or chemotherapy with Herceptin. The selected drug-resistant markers may represent the resistance not only to the chemotherapy but also to Herceptin.
In summary, our study has led to the identification of a list of important breast cancer proteins. The study also suggests that MS-based protein profiling may be an important tool in discovery of cancer biosignatures for tumor subtyping and prediction of treatment outcome. When sufficiently validated, some of these candidate protein markers could be used to improve breast cancer care. In addition, due to the heterogeneous and complex nature of the breast cancer tissue specimens, more refined methods need to be developed to maximize the protein identification to allow the capture of the best protein candidate markers for clinical use.
Diagonal linear discriminant analysis
Galectin-3-binding protein
Gene expression pattern analysis suite
Intermediate responders (>25% but ≤75% tumor regression)
K nearest neighbor
Mass spectrometry
Nonresponders, chemoresistant tumors (≤25% tumor regression)
Prediction analysis with microarrays
Pathological complete response, no residual cancer found at primary tumor site
Responders (>75% of tumor regression)
Self-organizing map
Support vector machines
Taxotere/Carboplatin/±Herceptin treatment
Triple negative breast tumors
Tumor regression rate.
The authors declare that they have no conflict of interests.
Grant support: The present study was supported by grant funds from NIH (NCI no. 1RO1 CA 093736-01A1), the Gonda Foundation, the Entertainment Industry Foundation (EIF)/Women's Cancer Research Fund, and the Friends of the Breast Program at UCLA.
The authors thank Jeffrey A. Gornbein (Department of Biomathematics, UCLA) for providing critical advice on statistical analysis.