Proteomic Profiling of Mammary Carcinomas ... - ACS Publications

Proteomic Profiling of Mammary Carcinomas Identifies C7orf24, a γ-Glutamyl Cyclotransferase, as a Potential Cancer Biomarker Pavel Gromov,†,‡ Irina Gromova,†,‡ Esbern Friis,†,§ Vera Timmermans-Wielenga,†,| Fritz Rank,†,| Ronald Simon,⊥ Guido Sauter,⊥ and Jose´ M. A. Moreira*,†,‡ Danish Centre for Translational Breast Cancer Research (DCTB), Strandboulevarden 49, DK-2100, Copenhagen, Denmark, Department of Proteomics in Cancer, Institute of Cancer Biology, Danish Cancer Society, Denmark, Department of Breast and Endocrine Surgery, Rigshospitalet, Denmark, Department of Pathology, the Centre of Diagnostic Investigations, Rigshospitalet, Denmark, and Department of Pathology, Diagnostic Center, University Medical Center Hamburg-Eppendorf, Hamburg, Germany Received February 23, 2010

Breast cancer is the leading cause of cancer deaths in women today and is the most common cancer (excluding skin cancers) among women in the Western world. Although cancers detected by screening mammography are significantly smaller than nonscreening ones, noninvasive biomarkers for detection of breast cancer as early as possible are an urgent need as the risk of recurrence and subsequent death is closely related to the stage of the disease at the time of primary surgery. A set of 123 primary breast tumors and matched normal tissue was analyzed by two-dimensional (2D) gel electrophoresis, and a novel protein, C7orf24, was identified as being upregulated in cancer cells. Protein expression levels of C7orf24 were evaluated by immunohistochemical assays to qualify deregulation of this protein. Analysis of C7orf24 expression showed up-regulation in 36.4 and 23.4% of cases present in the discovery sample set (123 samples) and in an independent large TMA validation data set (2197 samples) of clinically annotated breast cancer specimens, respectively. Survival analysis showed that C7orf24 overexpression defines a subgroup of breast tumors with poor clinical outcome. Up-regulation of C7orf24 was also found in other cancer types. Four of these were investigated in greater detail, and we found that a proportion of tumors (58% in cervical, 38% in lung, 72% in colon, and 46% in breast cancer) expressed C7orf24 at levels exceeding those seen in normal samples. The observed overexpression of this protein in different types of cancer suggests deregulation of C7orf24 to be a general event in epithelial carcinogenesis, indicating that this protein may play an important role in cancer cell biology and thus constitute a novel therapeutic target. Furthermore, as C7orf24 is externalized to the tissue extracellular fluid and can be detected in serum, this protein also represents a potential serological marker. Keywords: breast cancer • cancer biomarker • glutathione metabolism

Introduction In 2008, in Europe, there were an estimated 3.2 million new cases of cancer diagnosed (excluding nonmelanoma skin cancers) and 1.7 million deaths attributed to cancer. Breast cancer is by far the most frequent cancer in women (28.2% of all cancers) with an estimated 421 000 new cases in 2008, and it ranks second overall when both sexes are considered together.1 * To whom correspondence should be addressed. Jose´ M.A. Moreira, Department of Proteomics in Cancer, Institute of Cancer Biology and Danish Centre for Translational Breast Cancer Research (DCTB), Danish Cancer Society, Strandboulevarden 49, DK-2100 Copenhagen Ø, Denmark. E-mail: [email protected]. † Danish Centre for Translational Breast Cancer Research. ‡ Danish Cancer Society. § Department of Breast and Endocrine Surgery. | Centre of Diagnostic Investigations. ⊥ University Medical Center Hamburg-Eppendorf. 10.1021/pr100160u

 2010 American Chemical Society

Approximately 40% of patients presenting with lymph-node positive disease will experience a relapse. For patients with lymph-node negative disease, the 5-year recurrence rate is approximately 25%, suggesting that the risk of recurrence and subsequent death is closely related to the stage of the disease at the time of primary surgery. It is reasonable to assume that a decrease in the number of patients with advanced breast cancer will be followed by an equal decrease in breast cancerrelated mortality.2-9 Today, screening mammography is the only widely accepted method for early detection of breast cancer in asymptomatic women.10,11 Although screeningdetected cancers are significantly smaller than nonscreening detected ones, there is a pressing need to improve the efficiency and sensitivity of the method as it misses about 10% of the cases and gives a certain percentage of false positive results.12 Clearly, there is an urgent need to develop complementary diagnostic methods to detect breast cancer at a very early stage. Journal of Proteome Research 2010, 9, 3941–3953 3941 Published on Web 06/09/2010

research articles For the past years, our laboratory has carried out a systematic and comprehensive effort to search for markers for early detection and stratification of patients and novel targets for therapeutic intervention in breast cancer.13,14 Our research activities are part of long-term ongoing strategies at the Danish Centre for Translational Breast Cancer Research (DCTB) and have resulted in the identification of specific markers that can distinguish subtypes of breast cancer,15,16 markers that can categorize tumor cellular phenotypes,17 as well as candidate serological biomarkers.18 In this study, we employed twodimensional (2D) gel electrophoresis-based proteomic profiling of 123 primary breast cancers to discover protein markers with potential clinical usefulness. We identified a novel protein, C7orf24, that showed increased expression in a subset of tumors. C7orf24 was originally discovered as a 21-kDa protein involved in the release of cytochrome c from mitochondria in human leukemia U937 cells exposed to geranylgeraniol,19 indicative of the involvement of this protein in apoptosis. Furthermore, microarray-based studies have found C7orf24 to be differentially expressed and alternatively spliced in tumors.20,21 Recently, C7orf24 was identified as a γ-glutamyl cyclotransferase (GGCT), an enzyme that catalyzes the formation of 5-oxoproline (pyroglutamic acid) from γ-glutamyl dipeptides.22 Our results demonstrate that overexpression of C7orf24 is characteristic of a subset of breast tumors across histological types. We were also able to expand our analysis to other cancer types, such as cervical, colorectal, and lung cancer, and found that deregulation of C7orf24 is a recurrent theme in these cancer types.

Patients and Methods Patient Selection, Sample Collection, and Handling. Onehundred twenty-three women with primary, operable, highrisk invasive breast cancer were selected for this prospective study (Danish Center for Translational Breast Cancer Research - DCTB patient numbers 1-123; risk definition according to Danish Breast Cancer Cooperative group). Clinicopathological data for patients are given in Table 1. Patients had had no previous surgery of the breast and had not received preoperative treatment. Patients underwent mastectomy, which included axillary dissection in those cases with positive sentinel nodes. Mammary tissue specimens were collected from the Pathology Department at the University hospital immediately following surgery and were rapidly transported on ice to the Institute of Cancer Biology for further processing. On average 20-30 min elapsed from tissue acquisition to processing. Matched nonmalignant tissue was also collected whenever available. Specimens were divided into fractions with a fraction of each sample being stored at -80 °C for gel analysis. A similar tissue block was fixed in formalin and paraffin-embedded for immunohistochemistry (IHC) analysis. The project was approved by the Scientific and Ethical Committee of the Copenhagen and Frederiksberg Municipalities (KF 01-069/03). Cell Cultures and Bladder and Colon Samples. The human urothelial cancer cell line T24 was cultured according to ATCC’s guidelines and cellular extracts obtained by direct in plate lysis with lysis solution. Anonymous bladder cancer and colon cancer tissue specimens were collected at Skejby Hospital, Aarhus, Denmark and stored frozen until processed for gel electrophoresis. Preparation of Samples for Two-Dimensional Gel Electrophoresis. Twenty to thirty, 6-µm cryostat sections of frozen tissue were resuspended in 0.1 mL of lysis solution and were 3942

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Gromov et al. Table 1. Clinical Characteristics of Discovery Set (DCTBs) Specimens Number of patients Median age [range (years)] Median tumor size [range (mm)] Histologic type [n (%)] Ductal Lobular Other Histologic grade [n (%)] Grade 1 Grade 2 Grade 3 Unknown Lymph node status [n (%)] Positive Negative Unknown Estrogen status [n (%)] Positive Negative Progesterone status [n (%)] Positive Negative HER-2/neu status [n (%)] 0 1 2 3

123 62 (27-99) 28 (10-110) 100 (81) 19 (16) 4 (3) 27 (22) 57 (46) 37 (30) 2 (2) 94 (76) 26 (21) 3 (24) 99 (80) 24 (20) 75 (61) 48 (39) 25 35 34 29

(20) (28) (28) (24)

kept at -20 °C until used.23 The first and last sections of each sample were used for immunofluorescence analysis using keratin 19 antibodies as this epithelial marker is ubiquitously expressed by mammary epithelial cells. The availability of these pictures greatly facilitated the interpretation of the 2D PAGE studies as it gave a rough estimate of the ratio of epithelial cells to stromal tissue. The same procedure was applied to both nonmalignant and tumor tissue. 2D gels were analyzed using the PDQUEST software package version 8.0.1 (BioRad, Hercules, CA). Protein concentration was determined by the Bradford assay (Protein Assay Kit, Pierce, Thermo Fischer Scientific, San Diego, CA). Two-Dimensional Gel Electrophoresis. Tissue lysates were subjected to isoelectrofocusing (IEF) two-dimensional polyacrylamide electrophoresis (2D PAGE) as previously described.24 Between 5 and 40 µL of sample were applied to the first dimension, and at least 3 IEF gels were run for each sample. Proteins were visualized using a silver staining procedure compatible with mass spectrometry analysis.25 Protein Identification by Mass Spectrometry. Protein spots were excised from silver stained dry gels and the gel pieces were rehydrated in water. Gel pieces were detached from the cellophane film and cut into 1 mm2 pieces followed by “ingel” digestion as previously described.25 Samples were prepared for analysis by applying 2 µL of digested and extracted peptides on the surface of a 400/384 AnchorChip target plate (Bruker Daltonik, Germany) in duplicate, followed by cocrystallization with 0.35 µL of R-cyano-4-hydroxy-cinnamic acid (HCCA) (3 mg/mL in 66% acetonitrile, 0.5% TFA). Dry samples were washed twice by 2% TFA to remove contaminations. MALDITOF/TOF data were acquired using an Ultraflex III 200 timeof-flight mass spectrometer (Bruker Daltonik, Germany) equipped with a Smart beam laser and a LIFT-TOF/TOF unit. Data acquisition and data processing were performed by the Flex Control 3.0 and Flex Analysis 3.0 software (Bruker Daltonik,

Proteomic Profiling of Mammary Carcinomas Germany). All of the spectra were obtained using reflector positive mode with an acceleration voltage of 25 kV, reflector voltage of 26.38 kV and detection suppressed up to 450 Da. A total of 2000 shots in steps of 200 shots were added to one spectrum in the mass range of m/z 600-4000. Postacquisition two step calibration was automatically performed in FlexAnalysis using standard peptide calibration mixture (Bruker Daltonics, Germany) for external calibration followed by an additional internal calibration step to obtain better mass accuracies. Ubiquitous presented autodigested tryptic mass values visible in all the spectra were used for internal calibration. The background masses (matrix, metal adducts, tryptic peptides from keratins) were automatically subtracted from finally generated pick list and were excluded from the further analysis. For protein identification, peptide masses were transferred to the BioTools 3.2 interface (Bruker Daltonik, Germany) to search in the National Center for Biotechnology nonredundant NCBInr (20090905) database using an in-house MASCOT search engine (version 2.2, released 28.08.2009, Matrix Science Ltd.). No restriction on the protein molecular mass and taxonomy was applied as a first step. A number of fixed (acrylamide modified cystein, that is, propionamide/carbamidomethylation) and variable modifications (methionine oxidation and protein N-terminus acetylation) were included in the search parameters. The peptide tolerance did not exceed 30 ppm and a maximum of one trypsin missed cleavage was allowed. Protein identifications were considered to be confident when the protein score of the hit exceeded the threshold significance score of 65 (p < 0.05) and no less than 6 peptides were recognized. Often the peptides identified matched equally well to multiple database entries using the nonredundant NCBInr database that is why the second/final search was performed using the same parameters, but the UniProtKB/Swiss-Prot 55.3 (19372 human entries) database, and if the number and the sequence of the recognized peptides were identical to the first search, the Swiss-Prot accession number was assigned for the identified protein. Whenever it was possible or necessary, in case of low significance score for PMF, the MS/MS analysis by LIFT technology was additionally performed using the same search parameters where peptide/fragment tolerance did not exceed 30 ppm and 0.5 Da correspondently. Immunohistochemistry and Antibodies. Immunohistochemistry assays were performed using paraffin sections of human tissue samples essentially as described.26 Tissue blocks were placed in formalin fixative and subsequently paraffin-embedded for archival use. Five-µm sections were mounted on Super Frost Plus slides (Menzel-Gla¨ser, Braunschweig, Germany), baked at 60 °C for 60 min, deparaffinized, and rehydrated through graded alcohol rinses. Heat-induced antigen retrieval was performed by immersing the slides in Tris/EDTA pH 9.0 buffer (10 mM Tris, 1 mM EDTA) and microwaving in a 750 W microwave oven for 10 min. Nonspecific staining was blocked using 10% normal matched serum, 0.3% H2O2 in phosphatebuffered saline (PBS) buffer for 30 min. Antigen presence was visualized by incubation for 1 h with an appropriate primary antibody followed by detection with a suitable species-matched secondary antibody conjugated to a peroxidase complex for 30 min (Envision+ poly-HRP system; DAKO, Denmark). Color development was done using DAB+ Chromogen (DAKO). Slides were counterstained with hematoxylin. Standardization of the incubation and development times allowed accurate comparisons in all cases. The C7orf24-specific A255 rabbit polyclonal antibody was prepared by InBiolabs Ltd. (Tallinn, Estonia). The

research articles antibody was immunoaffinity purified against the immunizing peptide prior to use. Tissue Microarray and Immunohistochemical (IHC) Analysis. Construction of the breast prognosis tissue microarray (TMA) has been described in detail elsewhere.27 All other TMAs used in this study were acquired from Pantomics (Pantomics Inc., San Francisco, CA) and their description as well as the histopathology of tissue cores can be found at www.pantomics.com. Quantitative Assessment of Immunohistochemistry (IHC) Staining and Data Analysis. An automated cellular imaging system, ACIS III (DAKO), was used to digitize and quantify IHC staining intensity of tissue sections. The ACIS system is capable of simultaneously detecting levels of hue (color), saturation (density) and luminosity (darkness). By using the ACIS proprietary software, users can define threshold values for each of these parameters thus allowing the system to separately recognize brown pixels (positive immunostaining) and blue pixels (hematoxylin counterstain). For each tissue section, five distributed representative areas (Ø250 µm) of similar grade were defined and staining intensity values determined for each area.28 The overall section staining intensity was calculated as the mean value of the five areas. For survival analysis of TMAs, the ACIS III system was used to derive a staining score for each core. The digital images from scanned TMA sections were submitted to analysis by the TMA proprietary software module that is part of the ACIS III system. A staining score which is a function of staining intensity and percentage of cells showing immunoreactivity was generated in this manner for each core. Indirect Immunofluorescence Analysis. Five micrometer sections cut from paraffin blocks of breast tissue samples were mounted on Super Frost Plus slides (Menzel-Gla¨ser, Braunschweig, Germany), baked at 60 °C for 60 min, deparaffinised, and rehydrated through graded alcohol rinses. Heatinduced antigen retrieval was carried out as described above. Following antigen retrieval, sections were treated with ImageiT FX signal enhancer (Molecular Probes, Eugene, OR) to block nonspecific staining and subsequently incubated with the relevant primary antibodies (CK14 and C7orf24) at the appropriate dilution. Detection of immune complexes was done with species-specific secondary antibodies conjugated to Alexa Fluor 488 and Alexa Fluor 594 (Molecular Probes) and a directly conjugated antibody (CK19) to Alexa Fluor 633. Nuclear material was counterstained with TO-PRO-3. The sections were washed three times with cold phosphate-buffered saline (PBS) between incubations. Normal rabbit or mouse sera instead of primary antibody were used as a negative control. Cytokeratin antibodies (CK14 and CK19) were from NeoMarkers (Lab Vision Corporation, Fremont, CA). Sections were imaged using a Zeiss LSM510Meta confocal laser scanning microscope (Carl Zeiss MicroImaging Gmbh, Germany). 2D-Western Blot Analysis of Tissue Lysates. The specificity of the C7orf24 antibody was determined by 2D-PAGE immunoblotting using Western blots of tissue lysates prepared as previously described.24 Briefly, proteins were resolved by 2Dgel electrophoresis, blotted onto Hybond-C nitrocellulose membranes (Amersham Biosciences), and reacted with a C7orf24-specific rabbit polyclonal antibody (1:2000 A255 antibody) followed by detection of immune complexes with a horseradish peroxidase-labeled polymer (1:200) (Envision+ detection kit; DAKO). Blocking of antibody cross-reactivity was done using a protein-free blocking buffer (Thermo Fischer Scientific, Waltham, MA). Membranes were reversibly stained Journal of Proteome Research • Vol. 9, No. 8, 2010 3943

research articles with MemCode Reversible Protein Stain (Thermo Fischer Scientific, Waltham, MA) to match location of proteins in membrane with the Western blot signal. To identify the phosphorylation state of C7orf24, an aliquot of T24 cell lysate was treated for 30 min at 37 °C with 10 units of Shrimp Alkaline Phosphatase (Promega), and one aliquot was mock-treated prior to resolving by 2D gel electrophoresis. 2D-Western Blot Analysis of Serum samples. Peripheral blood samples were obtained with informed consent from all individuals. In the case of patient samples, blood was drawn preoperatively. The samples were collected into silicone-coated endotoxin-free tubes with EDTA as an anticoagulant (Vacutainers; Becton-Dickinson, Mountain View, CA). The sampling, handling, processing and storage of specimens were performed according to predefined Standard Operating Procedures. Sera were diluted 1:2 in 2D lysis buffer and samples subjected to IEF 2D PAGE as previously described.24 Between 20 and 40 µL of sample were applied to the first dimension. Gels were blotted onto Immobilon-PSQ polyvinylidene fluoride (PVDF) membranes (Millipore, Bedford, MA), and reacted with a C7orf24specific rabbit polyclonal antibody (1:500 A255 antibody) followed by detection of immune complexes with monoclonal mouse antirabbit immunoglobulins as second stage reagent followed by a horseradish peroxidase-labeled antimouse polymer (1:200) (Envision+ detection kit; DAKO). Blocking of antibody cross-reactivity was done using a protein-free blocking buffer (Thermo Fischer Scientific, Waltham, MA). Statistical Analysis. Kruskal-Wallis nonparametric test was used to assess significance of the association between C7orf24 expression and histopathological parameters (age and tumor size, histological type, BRE stage and grade, ER status, PR status, Her-2/neu status, and Ki67 score). Survival analysis was performed by the Cox proportional hazards model using the ACIS III intensity score as a continuous variable or by the Kaplan-Meier method, using various cut-points to dichotomize the intensity scores, with significance evaluated using the logrank test. In all analyses the significance level was set at 0.05. To correct for the influence of several variables (patient and tumor characteristics) we performed multivariate analysis using the Cox proportional hazards model. Statistical analysis of data was done with NCSS version 2007 (NCSS, Kaysville, UT).

Results 2D PAGE Analysis Revealed Up-Regulation of C7orf24 Protein in Breast Cancer Specimens. Fresh breast tumors and matched benign tissue specimens were analyzed by 2D PAGE. One of the proteins spots that showed frequent differential presence in tumor tissues as compared to nonmalignant samples was identified, in several samples, by mass spectrometry (MS)-based analysis as C7orf24 (γ-glutamyl cyclotransferase, Swiss-Prot O75223) (Figure 1). Some nonmalignant tissue samples exhibited a protein spot at the same location as C7orf24, however, MS analysis of several normal samples identified this protein spot as corresponding to a 21 kDa N-terminal fragment of annexin 2 (ANX2 fragment) (Figure 1A, red arrow) (Supporting Information). Gel image spot matching of C7orf24 and the ANX2 fragment showed that these two spots focused at the same position on the gel (compare Figure 1A with 1C, red arrows). Even though the ANX2 fragment was only present in some of the benign samples (compare Figure 1A with 1B), its overlapping with C7orf24 severely hindered the comparative analysis of C7orf24 expression in tumor tissues and matched benign tissue specimens based solely on 2D gel 3944


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Figure 1. Illustration of C7orf24 upregulation in breast carcinomas. Magnified sections of representative 2-D PAGE gels run with (A and B) lysates from nonmalignant tissue samples (Normal 34 and Normal 42, respectively) or (C) lysates from a tumor sample (Tumor 38) are shown. For each case, matched gels of externalized proteins from the same sample are also shown (D-F). Gels were visualized by silver nitrate staining. Arrowheads indicate the position of C7orf24 and ANX2 fragment (red arrowhead), and reference proteins (14-3-3ζ, β-actin, and Apolipoprotein D; white arrowheads) in the various gels.

protein profiling. As a consequence we decided, in a first phase, to evaluate how many breast tumors exhibited elevated relative levels of C7orf24 by comparison of 2D gels of tumor samples as nonmalignant samples displayed, at most, low levels of expression of the C7orf24/ANX2 fragment protein spot. In this manner we matched well-focused corresponding areas of 2D images and, as a result, could divide samples with good 2D gel protein profiles obtained from 118 breast cancer patients into three groups according to their relative levels of expression for C7orf24: low, medium and high. We found that in tumors, 17 samples out of 118 (14.4%) had no detectable expression, 58 specimens (49.2%) had low levels, 32 tumors (27.1%) showed medium levels and 11 (9.3%) showed high levels of C7orf24 protein expression. In conclusion, 43 out of the 118 (36.4%) lesions examined with good 2D gel protein profiles had medium to high levels of this protein, indicating that this protein may be up-regulated in mammary tumors. C7orf24 Protein Is Externalized into the Tissue Interstitial Fluid. Solid tumors display increased interstitial fluid pressure due to high vessel permeability, reduced lymphatic drainage, low perfusion, and increased number of cells in the region of the blood vessels.29,30 As a result, some of the proteins present in the tumor interstitial fluid (TIF) may reach the blood circulation trough the lymphatic system and/or blood vessels and hence represent potential serological markers for the early detection of cancer. We developed procedures to collect and characterize the protein composition of the tumor tissue interstitial fluid as this fluid is a potential source for the systematic search for potential serological biomarkers.18 Analysis of interstitial fluid 2D-gel protein profiles showed that C7orf24 was externalized to the extracellular fluid of tissues (Figure 1, compare panels C with F, red arrows) whereas ANX2 fragment was not (Figure 1, compare panels A with D, red arrows) confirming the MS-based analysis and indicating that C7orf24 may be present in serum and hence be a potential serologic biomarker of breast cancer. Determination of Antibody Specificity. To confirm the differential expression of C7orf24 in breast carcinomas, we

Proteomic Profiling of Mammary Carcinomas

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Figure 2. Determination of antibody specificity by 2D immunoblot analysis. Lysates of T24 cells were resolved by 2D PAGE (IEF). The resolved proteins were blotted onto a nitrocellulose membrane, reversibly stained with MemCode total protein stain (middle panel) and C7orf24 protein detected using a rabbit polyclonal antibodysA255. The identity of the protein spots detected were confirmed by matching the MemCode stained blot to a silver stained gel of the same sample (leftmost panel). (A) Two spots were detected in crude cellular lysates, a major spot corresponding to the unmodified form of the protein (black arrowhead) and a minor spot corresponding to a modified form of the protein (white arrowhead). (B) Lysates treated with alkaline phosphatase showed only the major unmodified form (black arrowhead).

generated peptide specific rabbit antibodies against this protein (Inbiolabs, Tallin, Estonia). One antibody, A255, showed exquisite specificity toward C7orf24 in 2D-PAGE immunoblot assays using both isoelectrofocusing and nonequilibrium pH gradient electrophoresis. The location of the protein spot(s) recognized by the antibody was mapped precisely by reversibly staining the membrane immunoblots and matching the stained blot with a silver stained gel of the same sample. As illustrated in Figure 2, for the T24 bladder epithelial cancer cell line, this antibody reacted only with C7orf24. Two polypeptide spots were detected: a major spot corresponding to the expected position of C7orf24 (Figure 2A, black arrow), and one minor, more acidic one (Figure 2A, white arrow). It was likely that some posttranslational modification was the cause of this double spot pattern. We investigated whether or not phosphorylation was responsible for the shift in apparent pI of the C7orf24 minor spot. Two-dimensional Western immunoblots of lysates treated with alkaline phosphatase prior to gel electrophoresis showed one single spot corresponding to the expected position of C7orf24 (Figure 2B, black arrow), indicating that phosphorylation caused the shift in the C7orf24 minor spot. Immunoblots of breast tumors that did not express ANX2 (Figure 3A) confirmed the specificity of the antibody toward C7orf24 and reiterated the pattern of expression of this protein in tissues, with a major spot corresponding to the unmodified form of the protein and a minor spot corresponding to the phosphorylated form of C7orf24 (Figure 3A, black and white arrows, respectively). We concluded that the A255 antibody specifically recognized C7orf24 and that this protein is generally present in two isoforms, a native and a phosphorylated state. IHC Analysis of C7orf24 Expression in Normal Mammary Epithelium and Breast Carcinomas. Having established the specificity of the antibody we proceeded to perform IHC analysis of paraffin-embedded tissue sections of normal breast samples (n ) 6) from reduction mammoplasty in patients without any clinical history of cancer. In all six cases normal breast epithelia showed weak immunoreactivity for C7orf24

Figure 3. Two-dimensional immunoblot analysis of C7orf24 expression pattern in tumor tissues. Lysates of fresh tissue specimens from (A) a breast invasive ductal carcinoma, (B) a bladder urothelial carcinoma, and (C) a colon carcinoma were resolved by 2D PAGE (IEF). The resolved proteins were blotted onto a PVDF membrane, and C7orf24 detected using the A255 antibody. In all cases we observed two spots corresponding to the unmodified form of the protein (black arrowheads) and a modified form of the protein (white arrowheads).

(illustrated in Figure 4A). C7orf24 immunoreactivity was restricted to the luminal cell layer (Figure 4A, black arrowhead), with myoepithelial cells being devoid of staining in normal mammary glands (Figure 4A, yellow arrowhead). IHC analysis of tumor samples showed that in a substantial number of specimens expression of C7orf24 in malignant cells was increased in comparison to benign tissue. This up-regulation is illustrated in Figures 4B and D (compare cells in benign tissue indicated by black arrowhead with malignant cells pointed out by red arrowhead). In some lesions, we could observe areas containing entrapped normal-looking ducts that displayed weaker C7orf24 staining (Figure 4B, black arrowhead) than that of adjacent malignant cells (Figure 4B, red arrowhead). Differential C7orf24 immunoreactivity with stronger cytoplasmic Journal of Proteome Research • Vol. 9, No. 8, 2010 3945

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Figure 4. Expression analysis of C7orf24 in formalin-fixed paraffin embedded breast tissue samples. (A) Immunohistochemical staining of C7orf24 protein in a normal breast tissue sample demonstrated the presence of the C7orf24 antigen in luminal epithelial cells with weak cytoplasmic and nuclear expression (black arrowhead). Yellow arrowhead points to a basal cell devoid of immunoreactivity for C7orf24. Scale bar, 50 µm. (B) Malignant cells showed stronger immunoreactivity (red arrowhead) than adjacent normal-looking ducts (black arrowhead), demonstrating up-regulation of this protein in tumor cells. Scale bar, 150 µm. (C) Tumor cells expressing highlevels of C7orf24 showed marked nuclear localization of this protein (red arrowhead). Scale bar, 50 µm. (D) We also observed upregulation of C7orf24 in some early lesions, such as DCIS, where carcinoma cells showed overexpression of C7orf24 (red arrowhead) in relation to adjacent normal-looking ducts (black arrowhead). Scale bar, 100 µm.

staining and strong, intermittent nuclear staining (Figure 4C, red arrowhead) of malignant cells was observed in patients bearing invasive lesions (Figure 4C) but also in some patients with carcinoma in situ lesions (Figure 4D) suggesting that upregulation of C7orf24, when it takes place, is most likely an early event in breast cancer progression. The staining phenotypes of normal glands and breast lesions were confirmed by four-color indirect immunofluorescence staining with C7orf24, cytokeratin 14 (CK14; a myoepithelial cell marker), and cytokeratin 19 (CK19; a luminal epithelial cell marker) antibodies (Figure 5). In normal breast tissue we observed complete colocalization between C7orf24 and CK19 cellular staining (Figure 5A) with no overlap with CK14 verifying the restricted expression of C7orf24 to luminal cells. Upregulation of C7orf24 is illustrated in Figure 5B (ductal hyperplasia). This sample presented very weak immunoreactivity for C7orf24 in normal luminal cells but increased expression in hyperplastic cells (transition point indicated by yellow arrowhead). Again, expression of C7orf24 was restricted to luminal cells. High Levels of C7orf24 Expression Occur in a Subset of Tumors Independently of Histopathology, Receptor Status, or Any Other Parameter that was Analyzed. Immunohistochemical analysis of paraffin-embedded tissue sections of those tissue blocks available from the tumor samples that were used for 2-D gel analysis (n ) 86), established that C7orf24 expression was increased in tumor samples compared to normal mammary tissue (Figure 6A; two-tailed t test P < 0.0001). 3946


Staining intensity scores mean in normal samples (n ) 6) was 54.1, whereas it was 60.2 in benign specimens collected from cancer patients (n ) 28), 69.4 in invasive ductal carcinomas (n ) 68), 66.7 in lobular carcinomas (n ) 14), and 69.1 in the remaining histopathology types (n ) 4). To assess the potential relevance of C7orf24 as a breast cancer marker and taking in consideration that having an independent sample data set is an important feature in the validation of biomarkers, we evaluated the expression of this protein in a breast cancer TMA containing 2197 tumors.27 We obtained interpretable staining results from 1137 out of 2197 (52%) tissue samples arrayed on the TMA. Lack of tumor cells and absence of cores were the main reasons for failure to obtain IHC data. Immunostaining intensities for C7orf24 varied greatly (Figure 6B) reminiscent of what we had observed for the DCTB sample set. A total of 266 out of the 1137 tumor samples (23.4%) displayed IHC staining intensities above the maximum values observed for normal control specimens (n ) 10) present on the TMA (Figure 6B, dotted line). Analysis of the IHC staining results from the TMA showed no statistically significant association (Kruskal-Wallis test) with any of the parameters we assessed, specifically: age and tumor size, histological type, BRE stage or grade, ER status, PR status, Her-2/neu status, and Ki67 score. Univariate hazard ratios were calculated by the Cox proportional hazards regression model using the ACIS III intensity score as a continuous variable. We found a trend in all tumors combined toward poor disease outcome in patients bearing


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Figure 5. Indirect four color immunofluorescence analysis of (A) a normal breast tissue section and (B) a hyperplastic lesion reacted with antibodies against C7orf24 (Alexa Fluor 488; green), cytokeratin 14 (CK14, Alexa Fluor 594; red channel), and cytokeratin 19 (CK19, Alexa Fluor 633; orange channel), and counterstained with the nuclear stain TO-PRO-3 (blue channel). Yellow arrowhead indicates beginning of region with hyperplastic cells. Scale bar, 50 µm.

Figure 6. Immunohistochemistry analysis of C7orf24 expression in breast samples. (A) DCTB discovery sample set was analyzed by IHC, including normal samples, benign specimens collected from cancer patients and invasive ductal carcinomas, lobular carcinomas, and other histopathology types. Mean intensity scores for each group are indicated by stippled lines. (B) Tukey outlier box and whisker plot of the ACIS staining scores for C7orf24 immunostaining of 2197 tumor samples present in a prospective TMA. Stippled line indicates maximum staining score obtained for a normal sample. Journal of Proteome Research • Vol. 9, No. 8, 2010 3947

research articles tumors with high levels of expression of C7orf24 but it did not reach statistical significance (P ) 0.0546). Dichotomization of intensity scores by use of the 10% (intensity score 80; HR) 0.95 [95%CI, 0.63-1.44]) and 90% (intensity score 116; HR) 0.97 [95%CI, 0.66-1.45]) percentiles showed that none of these cutoff points were statistically significant. Analyses of overall survival for specific histopathological types were performed on the results from the invasive ductal carcinoma (IDC) specimens (n ) 691), as no other histological type had sufficient data to perform this type of analysis. Univariate analysis using the ACIS III intensity score as a continuous variable showed there was a statistically significant trend toward expression of C7orf24 having prognostic value (P ) 0.042). Univariate survival analysis of samples with dichotomization of intensity scores by use of the 10% (intensity score 78; HR) 0.90 [95%CI, 0.58-1.41]) and 90% (intensity score 114; HR) 1.35 [95%CI, 0.86-2.11]) percentiles showed that these cutoff points were not statistically significant, but there was a trend toward poorer clinical outcome in patients with tumors exhibiting high levels of expression of C7orf24. Overexpression of C7orf24 is a Recurrent Event in Human Cancer. Previous studies have identified C7orf24 as being alternatively spliced in prostate cancer and differentially expressed in bladder cancer.20,31 To ascertain if this protein was also overexpressed in other tumor types, we examined the expression pattern of C7orf24 in normal human tissues and corresponding cancers. For this purpose we used the MNO661 multisystem TMA (Pantomics Inc., San Francisco, CA) containing 33 normal tissue types in duplicates (Table 2) and the MTU951 TMA containing 40 tumor types that covers most of the common benign, malignant and metastatic tumors originated from 27 anatomic sites (Figure 7). We found that C7orf24 was expressed in many of the normal tissues examined with weak to moderate immunoreactivity. In tumors, several different types of cancer including stomach, rectal, lung, ovarian, and prostate cancer showed up-regulation of C7orf24 (Figure 7). However, since only two to three samples were present for each different tumor type it was not possible to get realistic indications of the proportion of cases showing deregulation of C7orf2 for each tumor type. We used disease-specific TMAs (Pantomics) containing 150 cores (illustrated for colon cancer in Figure 8A) for each of the following four different cancers: colon (COC1501; including 75 cases of normal, reactive and cancerous tissues of the colon in duplicates), lung (LUC1501; including 75 cases of normal, inflammatory and common types of malignant lung tissues in duplicates), cervix (CXC1501; including 75 cases of uninvolved and neoplastic tissues of the cervix in duplicates) and breast (BRC1502; including 75 cases of normal, reactive, premalignant and malignant tissues of the breast in duplicates) to determine just how general deregulation of C7orf24 was. Of these four types of cancer, breast was used as a control, lung cancer as a cancer type that had already shown up-regulation in samples in the MTU951 TMA, and cervical and colon cancer as two types of cancer that had no cases with up-regulation of C7orf24 in the MTU951 TMA. Antibody dilutions were titrated in all cases so that normal tissue samples showed minimal immunoreactivity for C7orf24 (A255 antibody dilution 1:1200-1:1500), allowing us to better assess up-regulation of the protein (illustrated in Figure 8B, cf. panels 1 and 2, normal and cancerous samples, respectively). We found that in all four cancer types there was a proportion of tumors that expressed C7orf24 at levels exceeding those seen in normal tissue of the same organ (58% in cervical, 38% in 3948


Gromov et al. Table 2. Normal Human Tissue Expression Pattern of C7orf24 tissue

immunostaining

Adrenal gland Urinary bladder Bone marrow Eye Cerebellum Breast Cerebral cortex Fallopian tube Gastrointestinal tract Esophagus Stomach Small intestine Colon Rectum Heart Kidney Liver Lung Ovary Pancreas Parathyroid Pituitary gland Placenta Prostate Skin Spinal cord Spleen Striated muscle Testis Thymus Thyroid Tonsil Uterus cervix endometrium

cortical cells- moderate urothelial cells- weak/moderate n/da n/da granular and molecular layers- weak luminal epithelial cells- weak glial cells and neurons- weak n/da squamous epithelial cells- focal weak epithelium- weak/moderate epithelium- weak epithelium- weak epithelium- weak n/da renal tubules- moderate hepatocytes- weak alveoli- focal weak n/da n/da weak/moderate n/da n/da n/da n/da n/da focal weak myocytes, focal weak Leydig cells, weak n/da moderate squamous epithelial cells, weak squamous epithelia, weak epithelium, moderate

a Nondetectable under the conditions of the assay (antibody dilution 1:1000).

lung, 72% in colon, and 46% in breast cancer). We conclude that overexpression of C7orf24 is a common event in cancer.

Discussion Proteomic analysis of human breast carcinomas revealed a differentially expressed protein spot that was identified by MS as C7orf24, a γ-glutamyl cyclotransferase. We found that 43 out of the 118 (36.4%) lesions examined with good 2D gel protein profiles showed up-regulation of this protein, with 11 samples (9.3%) showing high levels of C7orf24 protein expression. To verify the deregulation of C7orf24 in tissues specimens directly, we generated rabbit polyclonal antibodies against this protein. One such antibody, A255, was highly specific for C7orf24 (Figure 2). We could thus determine that the protein was present in two forms, a major unmodified form and a minor modified form (Figure 2A; black and white arrowheads, respectively). Treatment of cellular lysates with alkaline phosphatase prior to gel electrophoresis eliminated the minor modified form of C7orf24 showing that this isoform was a phosphorylated form of the protein. The shift in pI was compatible with a single phosphorylation of the protein indicating that C7orf24 was present in a monophosphorylated form. This correlates well with previously published results, as large-scale phosphorylation analysis of mouse liver proteins identified C7orf24 as a phosphoprotein with a single phospho-


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Figure 7. Expression analysis of C7orf24 in normal tissues (red bars), corresponding cancer tissues (blue bars), and metastatic cancers (black bars) present in the MTU951 TMA, which contains 40 tumor types originated from 27 different anatomic sites.

rylation at Ser173.32 This site is conserved in humans, suggesting that the human C7orf24 protein is post-translationally modified in human tissues with a single phosphorylation at the orthologous residue Ser173. On the basis of previous studies, the expression levels of C7orf24 were expected to be relatively high in bladder and salivary gland,22,31 and kidney and liver.33 Our own IHC analysis showed that expression in normal urinary bladder, adrenal gland, stomach and kidney tissues was higher than in other major organs and tissues (Table 2). Yet even in the T24 bladder cancer cell line, which expresses high levels of this protein, we could not detect any variants of C7orf24 contrasting with previous studies that identified multiple isoforms of this protein in rat and human tissues.33-35 Having identified C7orf24 in breast cancer samples using a gel-based proteomics strategy, this protein was pursued for initial validation as a candidate biomarker. IHC-based analysis of C7orf24 expression in normal breast tissue showed that expression of this protein was restricted to luminal epithelial cells (Figures 4A and 5A) and confirmed overexpression of this protein in a subset of breast tumors (Figure 6A). We found also that upregulation of C7orf24 was an early event, as early lesions displayed increased levels of this protein (Figures 4D and 5B). Recently, an antibody-based comprehensive atlas of human protein expression patterns became publicly available through the Human Protein Atlas (HPA) program.36 This resource allows IHC-based evaluation of expression and localization of proteins of interest on 48 types of normal tissues, and 216 human tumors representing the 20 most common forms of human cancer. The HPA program reports the IHC based analysis of GGCT patterns of expression for two antibodies. The first antibody HPA020735 shows an immunostaining pattern in normal and tumor tissues that does not match our own data. The second antibody, HPA029914, however, has an almost identical pattern of staining to our own C7orf24 antibody, with differential expression in normal and malignant tissues. The antibody validation tests show the HPA020735 antibody to have “medium specificity (not more than two antigens with signal between 15% and 40%)” whereas the HPA029914 antibody has “high specificity (no other antigen with signal >15%)”. We applied this second, commercially available, polyclonal antibody - HPA029914 (Atlas Antibodies AB, Sweden) to a subset of our samples and found that it generated concordant results to our IHC analysis (data not shown) thus validating it. Overexpression of C7orf24 in a subset of tumors (23.4% of specimens examined), detected in the 123 mammary carci-

noma discovery sample set, was verified in a second independent data set present in a large TMA (Figure 6B). C7orf24 expression showed no statistically significant association with any of the parameters we assessed, specifically: age and tumor size, histological type, BRE stage or grade, ER status, PR status, Her-2/neu status, and Ki67 score. Although patients bearing IDCs with very high levels (>3-fold maximum normal levels) of C7orf24 had a poorer clinical outcome than patients with tumors showing low/moderate levels of this protein (P ) 0.0028) the small number of samples we had at hand displaying such abnormally high levels of the protein (n ) 20, corresponding to 3% of IDC cases) did not allow us to make a more conclusive analysis. Since C7orf24 encodes for GGCT, an enzyme in the γ-glutamyl cycle, it is likely that this protein is involved in glutathione homeostasis.22 Glutathione plays a key role in the pathophysiology of many different human diseases, including cancer.37,38 Glutathione has a pleiotropic effect on several key cellular processes related to cancer such as cell proliferation, apoptosis, and signaling, although definitive evidence for a cause and effect link between glutathione metabolism and cancer is still lacking. Several studies have found regulation of cellular glutathione levels to be correlated with disease outcome39,40 and resistance to treatment.41-43 Overexpression of C7orf24 was a common event in cancer. Of all the cancer types examined only in skin tumors, glial cell tumors, and lymphomas did we fail to detect any up-regulation of C7orf24 (Figure 7). In all other tumor types tested we found that a proportion of samples displayed increased expression of C7orf24 as compared to normal tissue. Significantly, C7orf24 was identified as 1 out of 46 genes that form a common cancer signature in a study performing large-scale integration of cancer microarray data that directly merged cancer/normal wholegenome microarray data sets to form an integrated training data set with 799 samples. These data sets span 21 tissue types including lung, breast, bladder, ovarian, pancreas, brain, prostate, uterus, colon, blood, kidney, uvula, salivary gland, thyroid gland, liver, skin, gastric tissue, myometrium, bone marrow, adrenal cortex and gastresophagus.21 Given that only a fraction of mammary carcinomas (20-40%) express C7orf24 at levels that are above those found in normal tissue and of these only a fraction (3-fold the levels found in normal samples), compounded by the fact that C7orf24 is expressed by a number of human normal tissues, one would not expect that its use as a sole biomarker will be feasible. Therefore, the most plausible Journal of Proteome Research • Vol. 9, No. 8, 2010 3949

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Gromov et al.

Figure 8. Expression analysis of C7orf24 in different cancer types. Representative C7orf24 immunostaining images of (A) a tissue microarray (TMA COC1501) and (B and C) tissue cores showing low and high ACIS score values (normal tissue core and tumor tissue core, respectively. Cores correspond to samples indicated in panel D with red circles). Scatter plots of ACIS intensity scores for IHC immunoreactivity in four disease-specific TMAs, illustrating deregulation of C7orf24 in (D) colon, (E) lung, (F) cervical, and (G) breast cancer.

course of action would be to incorporate C7orf24 into a panel of cancer biomarkers. In conclusion, this study demonstrates the differential expression of C7orf24 in breast tumors for the first time and further documents the potential use of C7orf24 as part of a universal cancer serum biomarker panel. We found C7orf24 to be externalized (Figure 1B) to the tumor tissue fluid indicating that it may be present in peripheral blood. In fact, the Proteomics Identifications Database (PRIDE, http://www.ebi. ac.uk/pride), reports the identification of C7orf24 in human plasma/serum in several data sets (http://www.ebi.ac.uk/ pride/searchSummary .do?queryTypeSelected)identification% 20accession%20number&identificationAccessionNumber)O75223; 3950


last accessed May 17, 2010). We examined the expression by 2D Western blot of C7orf24 in peripheral blood samples collected from three DCTB patients (DCTB numbers 20, 26, and 40) that showed moderate to strong immunoreactivity for C7orf24 in their tumors (Figure 9A-C). In one case, tumor 40, we could detect the presence of C7orf24 as one single spot (Figure 9G, arrowhead). No C7orf24 protein was detected in the peripheral blood samples of the healthy control subjects or in the two remaining patients (data not shown). Silver stained 2D PAGE gels of all serum samples, tumors as well as controls (Figure 9D-F and H-M, respectively), showed similar protein patterns for all samples. The small number of samples examined does not allow for further conclusions than confirm-

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Figure 9. Expression analysis of C7orf24 in human serum. Sera from three breast cancer patients (tumors 20, 26, and 40) with moderate to strong immunoreactivity for C7orf24 in corresponding tumor tissue (A-C), were subjected to IEF 2D PAGE (D-F) and analyzed by 2D Western blot for presence of C7orf24. In one case (G), tumor 40, we could detect the presence of C7orf24 as one single spot (arrowhead). Serum samples from six control subjects (H-M) were also examined but no C7orf24 protein could be detected. Scale bar, 100 µm.

ing the presence of C7orf24 in the peripheral blood of some patients in a potentially discriminatory manner. However, these findings are preliminary and require confirmation in larger studies. More in depth clinical validation studies are warranted to examine the potential for C7orf24 to serve as a candidate biomarker for cancer. Our data also suggests that C7orf24 may play a role in carcinogenic processes common to different cancer types and thus constitute a possible common therapeutic target warranting further studies into the role of this protein in cancer. Abbreviations: IHC, immunohistochemistry; 2D PAGE, twodimensional polyacrylamide gel electrophoresis; IEF, isoelectrofocusing.

Acknowledgment. We thank Kitt Christensen, Sofia Svensson, Signe Trentemøller, Lene Jørgensen, Hanne Nors, and Dorthe Holm for expert technical assistance. This work was supported by the Danish Cancer Society through the budget of the Institute of Cancer Biology and by grants from the John and Birthe Meyer Foundation, the Kai Lange and Gundhild Kai Lange Fond, the Saint Albans Church, the Lisa and Gudmund Jørgensens Fond, and the “Race against Breast Cancer”. The earmarked support of the Marketing Department at the Danish Cancer Society through their

fundraising activities appreciated.

on

behalf

of

DCTB

is

greatly

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topoisomerase inhibitor resistance and induces nonapoptotic cell death via mitochondrial-independent pathway. Cancer Res. 2006, 66 (11), 5772–80. (43) Li, W. S.; Lam, W. S.; Liu, K. C.; Wang, C. H.; Chang, H. C.; Jen, Y. C.; Hsu, Y. T.; Shivatare, S. S.; Jao, S. C. Overcoming the drug resistance in breast cancer cells by rational design of efficient glutathione S-transferase inhibitors. Org. Lett. 2010, 12 (1), 20–23.

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