also graded according to a modified version of the Scarff-Bloom-Richardson system (22). Exclusion criteria included patients who received previous partial or total resection in other hospitals, tumors that cannot be totally removed during operation, disease at stage IV, and occurrence of secondary malignancy during follow-up or metastatic disease within 90 d after surgery.
Of invasive carcinomas, 62% were associated with nodal disease. Of the 156 breast cancers, 90 (57.7%) were positive with ER, 68 (43.6%) with PR, and 35 (22.4%) with HER-2 overexpression, as seen on immunohistochemistry. At the time of diagnosis, 16.0%, 47.4%, and 36.6% of patients had tumors at stages I, II, and III, respectively. Median follow-up time for patients with invasive tumors was 44.33 mo (range, 3.16–97.58 mo). In 49 cases, disease relapsed and 19 patients died. Local and distant disease relapses that occurred during the 90-d postsurgery period were considered part of the primary event. Relapses after 90 days were considered new events. Relapses were dated and reviewed through the medical record. Disease-free survival was defined as the length of time after diagnosis to the first evidence of clinical recurrence or metastatic disease.
Immunohistochemistry of SCUBE2 expression.Breast cancer speci-men sections (4-Am thick) were dewaxed with xylene, rehydrated in graded concentrations of alcohol, treated with 3% H2O2for 30 min, washed with PBS, blocked with normal horse serum for 30 min, and incubated at 4jC overnight with anti-SCUBE2 antibody. Antibody binding was detected by using biotinylated antichicken antibody and horseradish peroxidase streptavidin (Vector) with 3,3¶-diaminobenzidine as chromogen (DAKO).
Hematoxylin was used as the counterstain. The immunostaining was considered positive when >10% of the tumor cells were immunoreactive.
Establishment of the MCF-7 breast cancer cell line stably expressing SCUBE2. The MCF-7 tetracycline-off (Tet-off) vector or MCF-7 Tet-off SCUBE2 cell lines were derived from the stable transfection of MCF-7 Tet-off cells (Clontech) with an empty pTRE2hyg plasmid (Clontech), a plasmid encoding the FLAG-tagged full-length (FL) or D4 mutant of human SCUBE2 (FLAG.SCUBE2-FL or FLAG.SCUBE2-D4), respectively. Stable cell clones were grown in the presence of 10Ag/mL doxycycline (to suppress SCUBE2 expression) and selected by resistance to G418 (100Ag/mL) and hygromycin (100 Ag/mL). Established cell lines were further verified by anti-FLAG Western blot analysis to assess doxycycline-responsive FLAG.SCUBE2 protein expression.
Immunoprecipitation, Western blotting, and flow cytometric analyses. Two days after transfection, cell lysates were clarified by centrifugation at 10,000 g for 20 min at 4jC. Samples underwent immunoprecipitation and then Western blot analysis as described (12). Cell surface expression of FLAG-tagged SCUBE2 was determined by anti-FLAG antibody staining and analyzed by a FACScan machine (Becton Dickinson).
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide cell proliferation assay.The effect of SCUBE2 on the proliferation of MCF-7 breast cancer cells was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Briefly, actively growing MCF-7 Tet-off vector and MCF-7 Tet-off SCUBE2-FL or SCUBE2-D4 stable cells
Table 1. Association of SCUBE2 protein expression and clinicopathologic characteristics and other biomarkers
Characteristics/markers Total SCUBE2 expression,n (%) m2test
Negative Positive P
Negative 66 35 (53.0) 31 (47.0)
Positive 90 35 (38.9) 55 (61.1)
PR status 0.02
Negative 88 47 (53.4) 41 (46.6)
Positive 68 23 (33.8) 45 (66.2)
HER-2/neu status 0.62
Negative 121 53 (43.8) 68 (56.2)
Positive 35 17 (48.6) 18 (51.4)
Recurrence <0.0001
Yes 49 35 (71.4) 14 (28.6)
No 107 35 (32.7) 72 (67.3)
SCUBE2, a Novel Prognostic Marker for Breast Cancer
were trypsinized and plated onto 96-well cell culture plates at 2,000 cells per well in 200AL complete media containing doxycycline. Doxycycline was removed from the medium on the next day to induce gene expression for various times. Each data point was performed in quadruplicate, and the results are presented as relative cell growth (%, meanF SD).
Tumorigenesis and growth of breast tumorsin vivo. Female athymic mice (8-wk-old nu/nu strain BALB/cAnN.Cg-Foxn1 nu/CrlNarl) were purchased from the National Laboratory Animal Center. Animals were allowed to acclimate to the new environment for 1 wk before being implanted with 0.5 mg of 17h-estradial 60-d release pellet (Innovative Research of America) s.c. on the dorsal side 1 d before tumor cell implantation to support the growth of the estrogen-dependent MCF-7 Tet-off cell-derived tumors. Before tumor cell implantation, mice were fed doxycycline-containing water (200Ag/mL), as described previously (23).
For tumor cell implantation, the MCF-7 Tet-off SCUBE2-FL or the MCF-7 Tet-off vector clone cells were harvested, washed with PBS, and resuspended in PBS. Then, 2 106cells in 0.2 mL of the mixture (50%
Matrigel, BD Bioscience) were injected into the mammary fat pads of female athymic mice. After tumor growth for 12 d, the mice were randomized to receive doxycycline-free or doxycycline-containing water to induce or suppress the expression of SCUBE2, respectively. Tumor size was measured twice a week by using digital calipers and calculated by length width height 0.5236 (in mm3). The experiments were terminated when the tumor size reached 800 mm3. Tumor growth in vivo was approximately exponential but varied slightly between animals. All surgical procedures followed protocols approved by the Institute Animal Care and
Luciferase activity assays. The bone morphogenetic protein (BMP)–
responsive luciferase reporter assay was performed as described previously (16).
In vitro digestion of SCUBE2 by purified recombinant matrix metalloproteinases.The FLAG-tagged SCUBE2 protein produced by HEK-293T cells was incubated with various recombinant matrix metalloprotei-nase (MMP; 500 ng; R&D Systems) in the absence or presence of a broad-spectrum MMP inhibitor (GM6001, 20 Amol/L, Merck) in a buffer containing 20 mmol/L Tris-HCl (pH 7.4)/5 mmol/L CaCl2/150 mmol/L NaCl/0.05% Brij-35 at 37jC for 2 h. The cleaved fragments were analyzed by probing with anti-FLAG (NH2 terminus) or anti–cysteine-rich repeats (COOH terminus) antibody, respectively.
Statistical analyses. Association of positive and negative SCUBE2 protein expressions and clinicopathologic variables of the carcinoma specimens was evaluated by m2 test. Kaplan-Meier survival curves were calculated with tumor recurrence/metastasis or death due to breast can-cer used as the end point. A log-rank test was used to calculate the disease-free survival, defined as the difference between SCUBE2-positive and SCUBE2-negative groups in time to recurrence. The Cox proportional hazard model was used to assess the effects of several possible prog-nostic factors with univariate analysis followed by multivariate analyses to identify independent prognostic factors for disease-free survival. All statistical tests were done with SPSS 10.0 (SPSS, Inc.). To compare the tumor growth rates of MCF-7 Tet-off SCUBE2-FL and MCF-7 Tet-off vector cells in animals, we estimated individual tumor volume at various times and then compared the growth rates by Student’s t test. A two-tailed P test was used in all analyses, and P < 0.05 was considered Figure 1. Kaplan-Meier disease-free survival curves for patients with breast cancer in terms of anti-SCUBE2–negative or anti-SCUBE2–positive status.A, all patients by negative (n = 70)or positive (n = 86)SCUBE2 status. B, patients with stage I disease by negative (n = 10)or positive (n = 15)SCUBE2 status. C, patients with stage II disease by negative (n = 34)or positive (n = 40)SCUBE2 status. D, patients with stage III disease by negative (n = 26)or positive (n = 31)SCUBE2 status.
Cancer Research
Results
Characterization of the anti–SCUBE2-specific antibody. To localize SCUBE2 protein expression in normal breast tissue or tumors, we first generated a polyclonal antibody specifically against SCUBE2. As shown in Supplementary Fig. S1A, this antibody recognized only SCUBE2 and did not cross-react with SCUBE1 or SCUBE3. Furthermore, we used this antibody for immunocyto-chemistry to detect SCUBE2 protein overexpressed in HEK-293T cells under formalin-fixed, paraffin-embedded conditions. As shown in Supplementary Fig. S1B, cells expressing SCUBE2 protein were positive for anti-SCUBE2 staining, whereas cells expressing SCUBE1 or SCUBE3 proteins were negative.
Expression of SCUBE2 protein in normal breast tissue.Using the anti-SCUBE2 polyclonal antibody, we found that endogenous SCUBE2 protein was expressed on the ductal epithelial or vascular endothelial cell surface of normal breast tissue (Supplementary Fig. S2A). Preincubation of the antibody with the corresponding immunogen peptide resulted in no staining, further showing the specificity of the anti-SCUBE2 staining in the breast tissues (Supplementary Fig. S2B).
Expression of SCUBE2 protein is correlated with favorable disease-free survival of breast cancer. To explore the role of SCUBE2 in breast tumor progression, we conducted a retrospec-tive study of SCUBE2 expression in 156 breast carcinoma biopsy samples. There are 86 (55.1%) of the primary tumors scored as positive for SCUBE2 (Supplementary Fig. S2D) and 70 cases (44.9%) were scored as negative for SCUBE2 (Supplementary Fig. S2C). To evaluate the potential contribution of SCUBE2 protein expression to prognosis of breast cancer, we compared clinicopathologic characteristics of cases scored as positive and negative for SCUBE2. As shown in Table 1, SCUBE2 protein expression had a significant negative association with tumor recurrence (P < 0.0001) and PR expression (P = 0.02) but is not associated with other characteristics.
The effect of SCUBE2 expression on disease-free survival was further analyzed by the Kaplan-Meier method. Survival analysis revealed a statistically significant relation between positive SCUBE2 protein expression and favorable disease-free survival in breast cancer patients (Fig. 1A). Patients were further stratified by initial tumor stage, but SCUBE2 protein expression remained significantly associated with favorable disease-free survival for patients with stage I (P = 0.0183), stage II (P = 0.0165), or stage III (P = 0.0007) disease (Fig. 1B–D).
Univariate analysis revealed a significant correlation between disease-free survival and SCUBE2 protein expression (P < 0.0001),
lymph node involvement (P = 0.02), advanced clinical stage (P = 0.0001), or PR status (P = 0.03) of breast cancer (Table 2).
Further multivariate analysis based on Cox proportional hazards models showed that clinical stage [hazard ratio (HR) 2.86, 95%
confidence interval (95% CI) 1.25–6.51] and positive SCUBE2 protein expression (HR 0.26, 95% CI 0.13–0.49) remained indepen-dent prognostic factors for disease-free survival (Table 2).
Overexpression of SCUBE2 protein suppresses proliferation of MCF-7 breast cancer cell line.Because the clinicopathologic association study implied that positive SCUBE2 protein expression was negatively correlated with tumor recurrence, we speculated that overexpression of SCUBE2 protein may lead to suppression of growth of breast tumors. To test the hypothesis, we first engineered stable MCF-7 breast cancer cell lines (MCF-7 Tet-off SCUBE2-FL clones) with the expression of FLAG.SCUBE2-FL protein under the control of an inducible promoter, the Tet-off promoter. In addition, the MCF-7 Tet-off vector clones containing stable integration of the empty expression vector were established as controls. Doxycyclin was removed from the medium to induce the expression of FLAG.SCUBE2-FL protein, determined by anti-FLAG Western blot analysis 1 to 5 days after doxycycline withdrawal. FLAG.SCUBE2-FL protein was readily expressed within 1 day, and expression peaked atf4 to 5 days after doxycycline removal (Fig. 2A). No induction of FLAG.SCUBE2-FL was observed in the presence of doxycycline in the MCF-7 Tet-off SCUBE2-FL clones (Fig. 2A) or in the control MCF-7 Tet-off Vector clones (data not shown).
To examine the effect of SCUBE2 overexpresssion on breast cancer cell growth, the MCF-7 Tet-off vector or MCF-7 Tet-off SCUBE2-FL stable cells were cultured in the presence or absence of doxycycline for 12 days to suppress or induce the expression of ectopic FLAG.SCUBE2-FL protein, respectively. Cell proliferation was then measured by MTT assay. Induction of ectopic SCUBE2-FL protein suppressed the growth of the MCF-7 Tet-off SCUBE2-FL clone cells in the absence of doxycycline (Fig. 2B). Furthermore, overexpression of the SCUBE2-D4 mutant protein, like the FL protein, suppressed the growth of the MCF-7 breast cancer cells (Supplementary Fig. S3). As a control, growth of MCF-7 Tet-off vector and MCF-7 Tet-off SCUBE2 cells did not differ on culture with doxycycline to block the expression of ectopic SCUBE2 protein (data not shown).
SCUBE2 represses tumor growth of MCF-7 cells in vivo.
Because SCUBE2 overexpression inhibited MCF-7 breast cancer cell growth in vitro, we next investigated breast tumor growth in vivo in nude mice. MCF-7 Tet-off vector or MCF-7 Tet-off SCUBE2-FL cells were injected into the mammary fat pads of nude
Table 2. Univariate and multivariate survival analysis by Cox proportional hazards models
Variables Univariate Multivariate
HR (95% CI) P HR (95% CI) P
SCUBE2 (positive versus negative) 0.25 (0.13–0.47) <0.0001 0.26 (0.13–0.49) <0.0001
Grade (1 and 2 versus 3) 1.39 (0.79–2.43) 0.26 1.12 (0.58–2.16) 0.74
Lymph node status (positive versus negative) 2.05 (1.10–3.82) 0.02 1.11 (0.46–2.68) 0.82
Stage (I and II versus III) 3.01 (1.71–5.30) 0.0001 2.86 (1.25–6.51) 0.01
ER (positive versus negative) 0.82 (0.46–1.44) 0.48 1.17 (0.53–2.56) 0.70
PR (positive versus negative) 0.51 (0.28–0.93) 0.03 0.73 (0.34–1.59) 0.43
HER-2/neu (positive versus negative) 1.39 (0.79–2.46) 0.26 1.02 (0.56–1.85) 0.95
SCUBE2, a Novel Prognostic Marker for Breast Cancer
mice that received estrogen pellets to promote the growth and development of breast tumors as described in Materials and Methods. After tumor growth for 12 days, the mice were fed doxycycline-free water to induce the expression of SCUBE2-FL.
Tumor growth from the MCF-7 Tet-off SCUBE2-FL cells in mice was markedly lower than that of tumors from control MCF-7 Tet-off vector cells (Fig. 2C). However, mice that continued to receive doxycycline-containing water to suppress the SCUBE2 induction showed no difference in tumor growth rate of MCF-7 Tet-off SCUBE2-FL or MCF-7 Tet-off vector cells (Supplementary Fig. S4). Together, these results showed that overexpression of SCUBE2 suppresses MCF-7 breast cancer cell growth both in vitro and in vivo.
SCUBE2 antagonizes BMP activity.Recent genetic study on zebrafish showed that BMP activity can be attenuated by the coexpression of SCUBE2 (24) and indicated that the COOH terminal cysteine-rich repeats and the CUBdomain are essential for zebrafish Scube2 function (24–26). In addition, BMPs are multifunctional growth factors that play important roles in normal cell differentiation and proliferation (27, 28) and have recently been implicated in promoting breast cancer cell proliferation (29, 30).
We then examined whether or not the COOH terminal fragment of SCUBE2 can interact with BMP2 protein or affect BMP signaling.
A series of FLAG-tagged SCUBE2 deletion constructs was first generated, including the SCUBE2-D4 deletion construct encoding for only the COOH terminal region (cysteine-rich repeat motif and CUBdomain) and two additional deletion mutants, SCUBE2-ty97 and SCUBE2-rw87, mimicking the ty97 and rw87 null mutant alleles (24–26), respectively, in the zebrafish Scube2 gene by removing various portions of COOH terminal domains (Fig. 3A).
HEK-293T cells were transfected with a Myc-tagged BMP2 expression plasmid alone or in combination with various FLAG-tagged SCUBE2 domain deletion constructs (Fig. 3B). Two days after transfection, cell lysates were subjected to immunoprecipita-tion with the anti-Myc monoclonal antibody, and the precipitates were analyzed by immunoblotting with anti-FLAG monoclonal antibody to determine protein interaction. Immunoprecipitation with anti-Myc antibody resulted in a specific coprecipitation of the SCUBE2-FL and SCUBE2-D4 deletion protein, but not SCUBE2-ty97 or SCUBE2-rw87 (Fig. 3B). These data suggest that SCUBE2 protein could indeed form a complex with BMP2 through its COOH terminal cysteine-rich repeats and CUBdomain.
To further examine whether the interaction between SCUBE2-D4 and BMP2 affected the signaling ability of BMP2, we performed a coculture assay in which the conditioned media derived from HEK-293T cells transfected with BMP2 alone or together with SCUBE2 deletion constructs (signaling cells) were added to the responding cells, HepG2 cells containing the BMP-responsive promoter luciferase reporter construct I-BRE-Luc (31). As expected, BMP2 alone produced by the signaling cells acted as a long-range signaling molecule by inducing an increase off6-fold in luciferase activity (Fig. 3C). Although the BMP2 protein coexpressed with SCUBE2-FL, SCUBE2-ty97, or SCUBE2-rw87 triggered the BMP-mediated transcriptional activation equally well, coexpression with the SCUBE2-D4 mutant resulted in marked attenuation of the BMP response (Fig. 3C).
Because the proteolytic processing of the large prepro precursor of BMP (proBMP) and its subsequent secretion into the extracellular space are the essential steps in the production of the biologically active form of BMP ligands, we then investigated
Figure 2. SCUBE2 overexpression suppresses MCF-7 breast cancer cell proliferationin vitro and breast tumor growth in vivo. A, induction of ectopic SCUBE2-FL protein in MCF-7 Tet-off SCUBE2-FL clone cells. MCF-7 Tet-off SCUBE2-FL cells were cultured in the medium without doxycycline [( )Dox] for 5 d, and the induction of ectopic NH2terminal FLAG-tagged SCUBE2-FL protein expression was determined by Western blot analysis with anti-FLAG antibody.
Anti–h-actin expression was used as a loading control. Double bands for the FLAG.SCUBE2-FL are due to the glycosylation of this protein (precursor and glycosylated, FL form). A limited proteoytic fragment is also observed after induction (cleaved).B, effect of SCUBE2 protein expression on MCF-7 breast cancer cell proliferation. The MCF-7 Tet-off vector and the MCF-7 Tet-off SCUBE2-FL stable cells were cultured in a medium without doxycycline [( )Dox]
to induce the expression of SCUBE2-FL protein. Cell proliferation was measured over the next 12 d by MTT assay. *,P < 0.01. C, induction of ectopic SCUBE2-FL protein reduces MCF-7 breast tumor growth in xenograft mouse model. The MCF-7 Tet-off vector or the MCF-7 Tet-off SCUBE2-FL stable cells were injected into nude mice to induce tumor formation. After tumor growth for 12 d and tumor development, the mice were divided into groups to continue to receive doxycycline or not [( )Dox]. Growth of the MCF-7 Tet-off vector or the MCF-7 Tet-off SCUBE2-FL cells was measured as a function of time in the absence of doxycycline.Points, mean tumor volumes; bars, SE. *, P < 0.01 (n = 13 for the MCF-7 Tet-off vector tumors and n = 14 for the MCF-7 Tet-off Cancer Research
the intracellular or extracellular environment. HEK-293T cells were transfected with plasmids expressing proBMP2 alone or in combination with various SCUBE2 deletion constructs. Western blot analysis of the cell lysates and conditioned media from these cultures revealed all deletion mutants with no effect on total proBMP2 synthesis (Fig. 3D); only the SCUBE2-D4 mutant potently suppressed the secretion of mature BMP2 into the culture medium.
Consistently, whereas overexpressed SCUBE2-FL or SCUBE2-ty97 proteins were secreted into the conditioned medium and properly targeted on the cell surface, the SCUBE2-D4 mutant was defective in membrane association and secretion (Fig. 4). Therefore, the
COOH terminal fragment represented by SCUBE2-D4, which binds BMP protein but without the NH2terminal region for membrane binding or secretion, acts to confine BMP protein within the cells, thus preventing its secretion and function (Fig. 3).
In vitro cleavage of SCUBE2 by purified recombinant MMP2.
To further clarify the physiologic significance of proteolytic processing of SCUBE2 in human breast cancers, we sought to identify the potential breast cancer–associated proteases that can cleave SCUBE2 protein in vitro. We initially undertook the candidate approach by examining the effect of MMPs (MMP1, MMP2, or MMP9) on the cleavage of SCUBE2, because these MMPs
Figure 3. The COOH terminal region of SCUBE2 binds BMP protein and acts as a BMP antagonist.A, domain organization of the SCUBE2 expression constructs used in this experiment. A FLAG epitope tag was added immediately after the signal peptide sequence at the NH2terminus for easy detection. Two deletion constructs, SCUBE2-ty97 and SCUBE2-rw87 mutant, were made by mimicking two null mutant alleles (ty97 or rw87) in the zebrafish Scube2 (24–26). FL, amino acids 1–1028;
D4, amino acids 664–1028; ty97, amino acids 1–659; rw87, amino acids 1–223; SP, signal peptide; E, EGF-like repeats; Cys-rich, cysteine-rich motifs;
CUB, CUB domain. B, interaction between SCUBE2 and BMP2. The BMP2 expression construct (Myc-tagged)was transfected alone or together with the expression plasmids encoding indicated FLAG-tagged SCUBE2 proteins in HEK-293T cells. Two days posttransfection, cell lysates underwent immunoprecipitation (IP)and Western blot analysis (WB)with antibodies as indicated to determine the protein interactions. proBMP2, precursor BMP2. C, coexpression of SCUBE2-D4 mutant attenuated the BMP2 signaling activity. Conditional media from signaling cells (HEK-293T)cotransfected with the BMP2 expression plasmid alone or in combination with various SCUBE2 deletion constructs as indicated were added to the responding cells (HepG2)that contained the BMP-responsive luciferase reporter plasmid I-BRE-luc (31).D, Western blot analysis of SCUBE2-D4 mutant inhibits BMP2 precursor processing into active, secreted molecule. HEK-293T cells were transfected
SCUBE2, a Novel Prognostic Marker for Breast Cancer
have been implicated in the proteolytic processes associated with breast cancer biology (32, 33). Recombinant NH2terminal FLAG-tagged SCUBE2-FL protein (FLAG.SCUBE2-FL) produced by HEK-293T cells was prepared and used as a substrate for in vitro reaction with purified recombinant MMP proteases. As shown in Supple-mentary Fig. S5, in vitro digestion of FLAG.SCUBE2-FL protein by MMP2, but not MMP1 or MMP9 (data not shown), induced the
have been implicated in the proteolytic processes associated with breast cancer biology (32, 33). Recombinant NH2terminal FLAG-tagged SCUBE2-FL protein (FLAG.SCUBE2-FL) produced by HEK-293T cells was prepared and used as a substrate for in vitro reaction with purified recombinant MMP proteases. As shown in Supple-mentary Fig. S5, in vitro digestion of FLAG.SCUBE2-FL protein by MMP2, but not MMP1 or MMP9 (data not shown), induced the