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[Cancer Research 64, 7346-7354, October 15, 2004]
© 2004 American Association for Cancer Research

Regular Articles

Progressive Loss of Syk and Abnormal Proliferation in Breast Cancer Cells

Maria Moroni1, Viatcheslav Soldatenkov4, Li Zhang4, Ying Zhang3, Gerald Stoica1, Edmund Gehan3, Banafsheh Rashidi2, Baljit Singh2, Metin Ozdemirli2,5 and Susette C. Mueller1

Departments of 1 Oncology, 2 Pathology, 3 Biostatistics, and 4 Radiation Medicine, Lombardi Comprehensive Cancer Center, Georgetown University Medical School; and 5 Department of Pathology, MedStar, Washington, District of Columbia


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The tumor suppressor gene Syk tyrosine kinase is absent or reduced in invasive breast cancer tissues and cell lines; its loss in breast tissues is linked to poor prognosis and metastasis. Also, evidence shows that in vitro Syk is involved in regulating proliferation. Here, we show by in situ hybridization on breast tissue sections that the loss of Syk expression is progressive during tumor development. Strikingly, Syk is already partially lost in normal epithelial tissue adjacent to the cancer lesion. In vivo, cell proliferation (as measured by the proliferative index Ki67) increased from normal to ductal carcinoma in situ to invasive, whereas Syk in situ staining in the same tissues decreased. In vitro, the presence of Syk was associated with reduced cell proliferation in an epidermal growth factor receptor-overexpressing breast cancer cell line, BT549, whereas changes in apoptosis were undetected. Concomitantly, the kinase activity of the proto-oncogene Src was reduced by ~30%. A 5-fold increase in abnormal mitoses was observed in the Syk-transfected cells compared with vector control. We propose that Syk is involved in the regulation of cell proliferation, possibly by controlling mechanisms of mitosis and cytokinesis via Src signal transduction pathway(s). Because of its progressive and early loss during tumor onset and development, monitoring of Syk loss in breast epithelial cells by noninvasive techniques such as ductal lavage may be a powerful tool for screening purposes.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Breast cancer is one of the most frequent and lethal types of cancers among women in Western countries. Syk tyrosine kinase, a widely expressed nonreceptor protein tyrosine kinase well characterized in blood cells, has been found recently to act as a tumor suppressor gene in epithelial breast cells (1, 2, 3) .

Syk contains two Src homology 2 (3) domains and multiple autophosphorylation sites. In blood cells, it couples immunoreceptors to transduction pathways regulating phagocytosis, cell differentiation, proliferation, adhesion, and motility (4, 5, 6, 7) . In B cells, it controls immunoreceptor-mediated calcium mobilization and phospholipase C activity, Ras/mitogen-activated protein kinase and phosphatidylinositol 3'-kinase/Akt survival pathway (8, 9, 10) . In hematopoietic cells, it mediates integrin signaling via both phosphorylation-dependent and -independent processes (11) . Besides blood cells, Syk is widely expressed in other cell types, such as epithelial cells, hepatocytes, fibroblasts, neuronal cells, and vascular endothelial cell lines, where it plays a variety of roles that are not yet completely understood (12) .

There are multiple lines of evidence that in breast tissue Syk plays the role of tumor suppressor. Syk mRNA is present in breast epithelial cells from normal tissues or cell lines but is reduced or absent in invasive breast carcinoma tissue and cell lines (1) . Transfection experiments showed that reintroduction of a wild-type Syk into highly aggressive breast cancer cells was sufficient to block tumor growth and experimental metastasis in vivo and anchorage-independent growth in vitro (1) . On the other hand, low tumorigenic cell lines containing endogenous Syk were more efficient in forming tumors that grew more aggressively in vivo after transfection with a Syk kinase-inactive mutant (1) . A recent finding also suggests that loss of Syk is linked to poor prognosis and metastasis (2) . The mechanisms of loss of Syk expression during breast cancer progression are just beginning to be investigated. In ~30% of invasive breast cancer, loss of Syk in cell lines and primary breast tumors can be explained by hypermethylation of the Syk gene (3) . Finally, a role for Syk in breast tumorigenesis is suggested by the link of allelic loss of the human Syk locus on chromosome 9q22 to lymph node metastasis of primary breast cancer (13) and by the development of mammary epithelial hyperplasia in mice where the negative Syk regulator c-Cbl was knocked out (14) .

Tumors are characterized by uncontrolled proliferation of the cells within a tissue, but it is still unclear with regard to Syk whether the driving force of tumor growth and metastasis is reduced cell death, increased cell proliferation, or none of the above. Syk is known to play roles in both apoptosis and cell proliferation. In B cells, natural killer cells, and DT40 cells, Syk promotes cell survival during oxidative stress, in some cases via activation of Akt (8 , 15, 16, 17) . In human eosinophils, Syk is also required to block apoptosis (18) , whereas in the case of B-cell receptor activation it positively mediates apoptosis (19) , and in DT40 irradiated with UVC it does not participate in the apoptotic process (17) . Concerning its role in cell proliferation, Syk stimulates growth factor-independent proliferation in hematopoietic cells by activating extracellular signal-regulated kinase 1/2 kinase (20) , as well as in endothelial cells and airway smooth muscle cells (21 , 22) . Both Syk and Src family members play a role in cell cycle regulation at the G2-M transition (23 , 24) . However, in contrast to the stimulation of cell proliferation of breast cancer cells mediated by Src (25 , 26) , Syk has been shown to block breast tumor cell growth both in vivo and in vitro (1) . In the latter study, experiments in which Syk was reintroduced into MDA-MB-435 cells did not reveal differences in either proliferation, detected by Ki67 staining, or apoptosis, detected by terminal deoxynucleotidyl transferase-mediated nick end labeling immunohistochemistry, in xenographed tumor tissue. However, abnormal mitoses with multipolar spindles were evident.

Here, we examine the relative levels of Syk mRNA expression in five different tissue types, normal, normal adjacent to cancer, hyperplasia, ductal carcinoma in situ (DCIS), and invasive tumor. A clear loss of Syk expression is evident during progression from normal to invasive tumor. Indeed, loss of Syk is already evident in adjacent benign tissue. Furthermore, we found a significant correlation of the loss of Syk in three of the tissue types, normal, DCIS and invasive tumor, with an increase in proliferative levels (Ki67). A role for Syk in cell proliferation in vitro is well documented in several cell lines, as mentioned above, and confirmed here for the first time in breast epithelial cells. Suppression of proliferation might occur via regulation of Src activity, because we found decreased Src tyrosine kinase activity in the presence of Syk. Therefore, Syk may be considered as a novel biomarker for tumor progression.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture.
The BT549 cell line was obtained by the Tissue Culture Shared Resources of the Lombardi Cancer Center and maintained in Falcon flasks (Becton Dickinson Labware, Bedford, MA) in Richter’s Improved MEM (Biofluids, Rockville, MD) supplemented with 10% fetal bovine serum, 2 mmol/L L-glutamine, 10 units/mL penicillin, and 10 µg/mL streptomycin (Life Technologies, Inc., Gaithersburg, MD). Wild-type full length human Syk was generated from pCA1F-Syk by a BamH1/HindIII restriction enzyme digest and subcloned into Bgll/HindIII digested pEGFP-C1 (Clontech, Palo Alto, CA). pEGFP-C1-Syk clones were identified by restriction enzyme digests and confirmed by automated DNA sequencing.

BT549 cells were transfected with pCA1F-Syk, pEGFP-Syk, or the vector controls using Fugene according to the manufacturer’s instructions (Roche Diagnostics Corp., Indianapolis, IN). Pools of pCA1F-Syk, enhanced green fluorescent protein-Syk, or their control vector-transfected cells were maintained in selection medium and used within 2 to 3 weeks (Improved MEM with 10% fetal calf serum containing 1 mg/mL G418). Enhanced green fluorescent protein-Syk transiently transfected for 2 days was also used in some experiments (fluorescence-activated cell sorted in Fig. 3BCitation or unselected in Fig. 6DCitation ). BT549 cells were transfected with enhanced green fluorescent protein or enhanced green fluorescent protein-Syk and harvested for fluorescence-activated cell sorter analysis at 2 days after transfection or after an additional 14 days of selection in 1 mg/mL G418. A duplicate transfection was performed so that we could compare in the same fluorescence-activated cell sorter experiment cells at 2 days after transfection with cells at day 14 of antibiotic selection, 16 days after transfection. Immediately after transfection (2 days), 23% of cells were expressing enhanced green fluorescent protein-Syk, and the relative mean fluorescence was 224 units. However, after 2 weeks of selection, the population of cells still expressing enhanced green fluorescent protein-Syk was ~8%, and the level of enhanced green fluorescent protein-Syk was lower (relative mean fluorescence, 100). Thus, Syk expression was gradually lost despite antibiotic selection, and cells with highest enhanced green fluorescent protein-expression were lost early.



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  		Fig. 3. Proliferation of BT549 in serum-containing medium. Cells were plated on uncoated 96-well plates in serum-containing medium, and cell proliferation was measured at different times (days) using an 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxy-phenyl)-2-(4-sulfonyl)-2H-tetrazolium assay (see Materials and Methods). Absorbance is directly proportional to the cell number. Measurements represent the mean values of six replicates. A, representative example of proliferation of pooled populations of BT549 cells stably transfected with pCA1F ({bullet}) or pCA1F-Syk ({circ}). B. Bt549 were transfected with EGFP ({blacksquare} and {blacktriangleup}) or with EGFP-Syk (open symbols) and sorted by flow cytometry. The top 50% to 90% expressing cells were used for the experiment. Parental cells are represented by {bullet}. For the others, each symbol represents an independent transfection experiment; bars, ±SE. (EGFP, enhanced green fluorescent protein)

 


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  		Fig. 6. Identification of Src kinase activity in BT549 cell extracts stably transfected with Syk or the vector control. A, total cell lysates prepared (see Materials and Methods) from BT549 cells stably transfected with pCA1F or with pCA1F-Syk from two independent transfections (as described in Fig. 3ACitation ) and that had been cultured for 48 hours in serum-containing medium. Proteins were separated by SDS-PAGE, transferred to nitrocellulose membrane, and probed first with antibody against the activated from of Src (Src-pY418) and subsequently with an antibody against actin to ensure equal amount of protein loading (at left). At right, the relative activity of Src (%) in BT549 transfected with pCA1F-Syk or the vector control was measured by scanning Western blots. Data are from three independent experiments. B. Western blot illustrates the level of endogenous Syk present in MCF-7 breast cancer cells compared with vector control and pCAIF-Syk stably transfected BT549 cells. The level of expression of Syk in transfected BT549 varied between ~1.4-fold (shown here) and 3- to 4-fold (data not shown) over the level in MCF-7 when equal protein loadings were evaluated by Western blotting with monoclonal anti-Syk antibody. C. The in vitro kinase activity of Src was determined. After lysis in radioimmunoprecipitation assay buffer of BT549/pCA1F or BT549/pCA1F-Syk cells, Src was immunoprecipitated from 0.5 mg of cellular lysate and then incubated under phosphorylation conditions with a synthetic substrate peptide (see Materials and Methods). The activity is reported as picomoles of {gamma}32 P-labeled phosphate incorporated in the peptide substratrum per minute (in vitro kinase activity). D. Total cell lysates were prepared from BT549 cells transiently transfected with EGFP or with EGFP-Syk for 2 days and cultured in serum-containing medium (see Materials and Methods). Proteins were separated by SDS-PAGE, transferred to nitrocellulose membrane, and probed with anti-Src and anti-Syk antibodies. Levels of Src protein were approximately equal in control EGFP or EGFP-Syk transfected cells (EGFP-Syk transfectants were 0.9-fold of EGFP control transfectants). MCF-7 cells had 0.4-fold less Src protein than BT549 control cells. EGFP-Syk protein expression was ~7.8-fold higher than MCF-7 endogenous Syk levels representing ~23% cells transiently transfected in the total population (Blot: anti-Syk, Src). The in vitro kinase activity of Src was determined in these transiently transfected cells and MCF-7 cells after immunoprecipitation as described above (at right). BT549/EGFP-Syk1 and BT549/EGFP-Syk2 represent two independent transfections of EGFP-Syk (in vitro kinase activity). (EGFP, enhanced green fluorescent protein)

 
Cell Death Assays.
Surface expression of phosphatidyl serine was determined by Annexin V staining (Trevigen, Gaithersburg, MD) as described previously (27) . Briefly, BT549 cells (2 to 5 x 105) were harvested, washed in PBS, and exposed to FITC-labeled Annexin V. To exclude staining of phosphatidylserine on the inner surface of the cell membrane, indicative of cytoplasmic membrane disintegration, cells were counterstained with propidium iodide. FITC-Annexin V and propidium iodide staining was determined using a FACStar Plus (Becton Dickinson, Franklin Lakes, NJ). Apoptotic cells were defined as FITC positive and propidium iodide negative.

Cell Sorting.
BT549 cells transfected with pEGFP-Syk or pEGFP were suspended in PBS and sorted by fluorescent activated cell sorting (FACStar Plus, Becton Dickinson) for pEGFP fluorescence as described in the text.

Cell Proliferation Assay.
Syk-transfected BT549 cells and vector controls were plated at a density of 1 x 103/well in 96-well plates and grown in serum-containing medium in the presence of G418 (1 mg/mL). Cells were then incubated at 37°C 5% CO2 for the times indicated. Cell proliferation was measured using a 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxy-phenyl)-2-(4-sulfonyl)-2H-tetrazolium assay (CellTiter 96 AQueous, Promega, Madison, WI). The assay was performed according to the manufacturer’s instructions. Briefly, at specified time points, 20 µL of 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxy-phenyl)-2-(4-sulfonyl)-2H-tetrazolium reagent was added to 100 µL of sample in a 96-well plate and incubated at 37°C for 3 hours. Absorbance at 490 nm was measured using a 96-well plate reader. All of the measurements were performed at least in four replicates.

Preparation of Whole Cell Lysates and Western Blot Analysis.
Equivalent numbers of cells were suspended in Lysis Buffer [50 mmol/L Tris-HCl (pH 8.0), 150 mmol/L NaCl, 40 mmol/L ß-glycerophosphate, 1% NP40, 50 mmol/L NaF, 1 mmol/L sodium phosphopyruvate, 1 mmol/L EGTA, 1 mmol/L EDTA, 1 mmol/L sodium vanadate, and 1 tablet/10 ml of protease inhibitors; Complete EDTA-free, Roche Diagnostics GmbH, Mannheim, Germany]. Cell lysates were kept on ice for 10 minutes followed by microcentrifugation at 10,000 x g for 10 minutes at 4°C. Supernatants were directly solubilized with SDS sample buffer [2% SDS, 62.5 mmol/L Tris-HCl (pH 6.8), 10% glycerol, 5% 2-mercaptoethanol, and 0.001% bromphenol blue] and boiled for 3 minutes.

Whole cell lysates were separated by SDS-PAGE and transferred to nitrocellulose membranes. Membranes were blocked with 1% bovine serum albumen in Tris-buffered saline-Tween [150 mmol/L NaCl and 10 mmol/L Tris-HCl (pH 7.4) containing 0.1% Tween 20] for 1 hour at room temperature and incubated with anti-Src-pY418 antibody (Biosource, Camarillo, CA) diluted in 1% bovine serum albumen in Tris-buffered saline-Tween overnight at 4°C. Membranes were washed and incubated with horseradish peroxidase-conjugated secondary antibodies in 1% bovine serum albumen in Tris-buffered saline-Tween for 30 minutes at room temperature. After washing, bands were visualized by enhanced chemiluminescence assays. Membranes were reprobed with antiactin antibodies to ensure equal amount of protein loading.

In vitro Kinase Assay.
Src kinase assay was performed using a kit (Upstate Biotech, Lake Placid, NY). Briefly, Src was immunoprecipitated from whole cell lysates, and its activity was measured in an in vitro kinase assay using a substrate peptide specific for Src and [{gamma}32P]ATP. The [{gamma}32P] incorporated into the substrate was quantitatively measured using a scintillation counter, and the enzymatic activity was expressed as picomoles of [{gamma}32P] incorporated into the substrate per minute.

Immunostaining.
One-hundred and thirteen formalin-fixed, paraffin-embedded breast tissue samples were obtained from the Lombardi Cancer Center Histopathology and Tissue Shared Resource. The samples were composed of the following groups: (1) histologically normal tissues from healthy individuals (n = 18), (2) tissues from patients diagnosed with DCIS (n = 43), and (3) tissues from patients with DCIS and invasive lesions (n = 52). The size of the tumors ranged between 0.1 and 5.5 cm. Formalin-fixed, paraffin-embedded tissue sections were stained with antibodies against Ki67 and Syk.

Briefly, 5-µm thick tissue sections were deparaffinized, rehydrated, and subjected to antigen retrieval by boiling in 10 mmol/L sodium citrate buffer (Zymed, San Francisco, CA). For Syk immunostaining, the rabbit EnVision Plus kit was used (DakoCytomation, Carpinteria, CA); the slides were incubated at 4°C overnight with the primary antibody (Cell Signaling, Beverly, MA) diluted 1:50. Slides were stained with 3,3'-diaminobenzidine, counterstained with H&E, and mounted. For Ki67 staining (Coulter Immunotech diluted 1:50), the Biogenex supersensitive kit was used. Briefly, after antigen retrieval in citrate buffer, tissue endogenous peroxidase activity was blocked with a peroxidase block. Slides were incubated with primary antibody for 45 minutes then with biotinylated secondary antibody, and finally with horseradish peroxidase-streptavidin complex. Slides were stained with 3,3'-diaminobenzidine, counterstained, and mounted.

For {alpha}-tubulin staining, BT549 cells were seeded onto glass coverslips. After 6 days, cells were fixed in methanol for 5 minutes at –20°C and then washed in PBS/0.1% Triton X-100. To block nonspecific binding, the coverslips were incubated in blocking solution (PBS containing 0.1% gelatin and 10% normal donkey serum) for 15 minutes followed by a 15-minute incubation in blocking solution containing a monoclonal anti {alpha}-tubulin (Sigma, St. Louis, MO) antibody (1:2,000 dilution) and sequential washing with PBS/0.1% Triton X-100. Cells were then incubated for 15 minutes at room temperature in the presence of blocking solution containing a FITC-labeled rabbit antimouse IgG (Molecular Probes). At the end of the incubation, coverslips were washed three times in PBS/0.1% Triton X-100, rinsed in PBS, and finally mounted with the Prolong Antifade mounting medium (Molecular Probes, Eugene, OR). Cells were viewed using an Olympus Fluoview confocal fluorescent scanning microscope equipped with a 60x/1.4 N.A. lens. The images were displayed with the accompanying software.

In situ Hybridization.
Formalin-fixed, paraffin-embedded tissue sections were rehydrated, immersed in HCl 0.2 N for 10 minutes, and digested with 10 µg/mL proteinase K for 30 minutes. Syk mRNA was allowed to hybridize with 100 ng of digoxigenin-labeled antisense probe overnight at 70°C (1) . Slides were then washed at 42°C in SSC (progressively 2x SSC, 1x SSC, and 0.1x SSC) and immunostained with an antibody against digoxigenin conjugated to alkaline phosphatase (DIG Wash and Block buffer set, 1 585 762, Roche). The signal was visualized by the nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate color reaction (DAKO) and slides counterstained with methyl green.

Statistical Methods.
A major objective of this study was to evaluate how Syk mRNA expression levels changed among the five kinds of tissues (normal, normal adjacent, hyperplasia, DCIS, and invasive). Similarly, an objective was to test whether Syk mRNA correlated with proliferation (measured as Ki67 immunostaining). The intensity of the expression levels was rated on a scale from 0 (no staining) to 4 (excess staining). Our hypothesis is that the mean expression level for Syk mRNA staining decreases with increasing invasiveness of tumor, so a linear statistical model, Y = a + bx, was fit to the data. Here X represented the type of tissue [normal (mammoplasty) = 1, normal adjacent = 1.5, hyperplasia = 2, DCIS = 3, and invasive = 4], Y = intensity level of the tissue (scored from 0 to 4), a and b are fitted values by least squares, and a is the intersect and b is the slope. For the data in Fig. 2Citation , the equation determined by the squares fitting was Y = 3.3588 – 0.5215X, indicating a highly statistically significant (P < 0.0001) negative slope. That is, as invasiveness of tissue increases, expression levels of Syk mRNA decreased.



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  		Fig. 2. Summary of Syk mRNA expression in human breast tissues. The intensity of the staining (blue-black precipitate) was evaluated by two independent pathologists and a score ranging from 0/1 (background) to 4 (very strong) was assigned to each tissue component (normal, hyperplasia, DCIS, and invasive) present on a slide. A plot of the means was generated for each tissue type, normal epithelium from reduction mammoplasty (Mammoplasty), normal epithelium adjacent to DCIS or invasive (Normal adjacent), hyperplasia, DCIS, and invasive tumor lesions (Invasive); Bars, ±SE.

 
Similarly, linear relationships were fit to the subset of data in Fig. 4BCitation relating intensity of staining to Syk (in situ) and proliferation (determined in adjacent sections by Ki67 staining). Here X represented the type of tissue (normal = 1, DCIS = 2, and invasive = 3). For the Syk (in situ) data, the equation obtained was Y = 3.7252 – 0.8620X, indicating a significantly negative slope (P < 0.0001). For proliferation (Ki67), the equation determined by least squares was Y = 0.4762 + 0.7435X, indicating a highly significant positive slope (P < 0.0001). The SE of the slope for the Syk in situ data was 0.1115 and that for the Ki67 data was 0.0776, whereas that for the data in Fig. 2Citation was 0.0698.



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  		Fig. 4. Inverse relation between Syk mRNA expression and cell proliferation in human breast tissue samples. A. Formalin-fixed, paraffin-embedded tissue sections were subjected to in situ hybridization with a probe against Syk mRNA (Syk). Adjacent sections (Ki67) were stained for the mitotic marker Ki67 (brown precipitate) and counterstained with hemotoxylin. In many cases, areas with higher expression of Syk mRNA showed lower mitotic index. Scale bar = 100 µm. B. The means of the relative levels of Syk mRNA and percentage of Ki67-positive cells are shown. Tissues stained with Ki67 were assigned a score from 1 to 4 (<5% positive nuclei = 1; 5% to 10% = 2; 11% to 20% = 3; >20% = 4). Sections containing normal, DCIS, and invasive tissues were analyzed. In situ slides were rated for each tissue as described in Materials and Methods. These slides were a subset of the total number used for analysis in Fig. 2Citation . The percentage of positive Ki67 nuclei were judged by the pathologist for each tissue type in the adjacent sections. C. Pearson rank correlation coefficients for pair-wise comparison of Ki67 versus Syk for normal versus normal, DCIS versus DCIS, or invasive versus invasive tissues.

 
Another objective of this study was to evaluate the relationship between Syk immunostaining and proliferation (Ki67). It was possible to evaluate this relationship in 44 normal, 36 DCIS, and 25 invasive tumors where paired observations of Syk expression and Ki67 were available. Rank correlation coefficients (r) were calculated, and the tests of the null hypothesis that the true correlation was zero were performed for each of the three types of tissues. The results are given in Fig. 4CCitation .

All of the data analyses were performed in SAS 8.0 (SAS Institute, Inc., Cary, NC).


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Expression of Syk mRNA in Human Breast Tissues.
Syk is lost in invasive breast cancer. However, it is still unknown whether the loss of Syk is a feature of the biology of invasive cancer or whether it might also play a role in the formation of noninvasive lesions such as carcinoma in situ and hyperplasia. To answer this question, we analyzed Syk mRNA expression in samples from 113 subjects, 18 of which were from mammoplasty reduction (no tumor lesions), 43 had only DCIS with or without hyperplasia, and 52 had also invasive components. Expression of Syk mRNA was analyzed by in situ hybridization with a digoxigenin-labeled probe conjugated to alkaline phosphatase (1) . Two independent pathologists assigned a score ranging from 0/1 (background) up to 4 (strongest) to the intensity of the signal, a blue-black insoluble precipitate, for each component present on the tissue slides.

Fig. 1Citation illustrates in situ staining for spleen and normal breast using the antisense RNA probe to detect Syk (Fig. 1ACitation , antisense). The sense probe was negative (Fig. 1ACitation , sense). The results of in situ staining of normal, DCIS, and invasive tumors are illustrated in Fig. 1BCitation and summarized in Fig. 2Citation . There is clear visual evidence of decreasing mean intensity levels as the invasiveness of tissues increased, which was confirmed statistically by fitting a straight line. The negative slope (–0.5215) was highly statistically significant (P < 0.0001; details in Statistical Methods). Very interestingly, the mean expressions in mammoplasty reduction specimens, 3.08 (SE = 0.11), were significantly higher than in normal tissues adjacent to cancer lesions, 2.57 (SE = 0.13), and the difference was highly statistically significant (P < 0.0004).



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  		Fig. 1. In situ detection of Syk mRNA expression in paraffin tissue sections. Breast tissue sections 5-µm thick were hybridized with a digoxygenin-labeled probe against Syk mRNA as described in Materials and Methods and a blue-black reaction product visualized. A. Paraffin sections of normal breast or spleen were hybridized with sense (negative control) or antisense Syk probes. Scale bar = 250 µm. B. Examples of normal tissue adjacent to a cancer lesion (normal), DCIS, and invasive component (invasive) are shown; the scores assigned for these examples were 3, 2, and 1, respectively. Scale bar = 100 µm.

 
Syk and In vitro Proliferation.
A correct cross-talk maintained by highly regulated enzymatic activities between external stimuli and internal signal transduction pathways is fundamental to the proper function of the cell and its interaction with the surrounding elements. To determine whether the loss of Syk during the progression of cancer might be involved in the dysregulation of cell proliferation, we examined the proliferation rate of BT549 cells stably transfected with Syk and compared it with BT549 transfected with the vector control. Detection of cell proliferation was through measurement of cellular metabolic activity via a spectrophotometric assay. Cells were seeded in 96-well plates in the presence of serum-containing medium, and cell proliferation was measured at different time points (days). Four independent experiments were run for each experiment and up to two independently transfected BT549-Syk pooled populations were used. A small but consistent difference between Syk-overexpressing cells and the control was seen for each pooled population in every experiment. A representative experiment is shown in Fig. 3ACitation . Although the difference in proliferation rate between Syk-overexpressing cells and the vector control was not striking (~25% of growth inhibition), the constant and reproducible nature of the result led us to wonder whether the level of expression of Syk in the stably transfected pooled populations was uniform. Surprisingly, although Western blots indicated strong expression of Syk, immunohistochemical staining of stable pooled populations with a specific antibody against Syk showed that only a very low percentage of cells was detectably expressing Syk protein, although 100% of the cells were resistant to the selection agent (data not shown). Western blots of Syk-expressing cells had been performed on a weekly basis and showed a continual decline in Syk expression with time after stable selection. This is not surprising given that Syk-negative cells appear to have a growth advantage. Therefore, a different type of strategy was used to ensure proper selection of Syk-expressing cells to confirm an effect of Syk upon proliferation. BT549s were transfected with a plasmid coding for either enhanced green fluorescent protein or enhanced green fluorescent protein-tagged Syk. A total of five independent transfections were done. Forty-eight hours after transfection, enhanced green fluorescent protein- or enhanced green fluorescent protein-Syk-positive cells were selected by flow cytometry. For each transfection experiment, the top 50% expressing cells were immediately seeded in serum-containing medium in 96-well plates and used for the cell proliferation assays as described above. The mean enhanced green fluorescent protein fluorescence signal for the pooled populations of enhanced green fluorescent protein-Syk and enhanced green fluorescent protein-expressing cells was the same. In the case of cells sorted for enhanced green fluorescent protein-Syk expression, a much faster and more dramatic effect on the proliferation rate was seen compared with the previous experiments using unsorted cell populations. After 5 days of culture, BT549 cells expressing enhanced green fluorescent protein-Syk already showed ~65% of inhibition of growth with respect to the matched controls and parental cells, thus confirming for BT549 the role of Syk in cell proliferation (Fig. 3B)Citation .

Relationship between Syk Expression and Ki67 Immunostaining in Breast Tissues.
We next studied the correlation of Syk mRNA expression and cell proliferation in vivo. Forty four among all of the cases stained by in situ hybridization were randomly chosen, and the adjacent sections were immunostained with an antibody against the proliferation marker antigen Ki67 (Fig. 4A)Citation . Results are shown in Fig. 4BCitation . Because of their different biology, normal tissue, noninvasive DCIS tumor lesions, and invasive cancer lesions were considered as different categories and were analyzed separately.

As is visually evident in Fig. 4BCitation , intensity of Syk mRNA decreases, whereas Ki67 proliferation increases with invasiveness of tumor. This has been confirmed statistically by fitting a linear relationship between intensity of expression levels to invasiveness of tissue that resulted in the significant negative slope (–0.8620) for Syk (in situ; P < 0.001) and a positive slope (0.7435) for Ki67 proliferation (P < 0.0001). Hence, there was strong statistical evidence of an approximate linear trend for increasing Ki67 levels and decreasing Syk mRNA levels from normal to invasive tissues, even in this subset of data.

Fig. 4CCitation gives the rank correlation coefficients (r) of Ki67 staining and Syk in situ levels, and the P of the test of H0: r = 0 for each type of tissue (normal versus normal, DCIS versus DCIS and invasive versus invasive). None of the Pearson correlation coefficients were statistically significantly different from 0. Hence, no significant association was detected between Ki67 and Syk in situ within each of the tissue types from this data set. That is, although strong differences were detected when tissue categories were compared (i.e., normal versus invasive), comparison of slides within a tissue type (i.e., invasive versus invasive) showed no statistical differences. Thus, within a particular category such as hyperplasia, Syk levels and proliferations levels were similar; however, they were markedly different from tissue type to tissue type.

Mitotic Figures.
We have shown previously that malignant cells transfected with Syk and injected in mice grew tumors at a slower rate and were characterized by a high number of multipolar spindles (1) . However, it is not uncommon for cancer cells to have more than two spindle poles per cell. To confirm whether aberrant mitosis was the cause of slower tumor cell proliferation, we used BT549 cells transfected with Syk or the plasmid control and determined the cellular mitotic index and the number of multipolar spindle profiles. BT549 cells grown in serum-containing medium for 6 days were then EtOH-fixed at –20°C and stained with antibodies against {alpha}-tubulin (Fig. 5A)Citation . About 1,000 cells per cell line were examined, and mitotic cells with established spindles poles were counted. The mitotic index of Syk-transfected BT549 was 70% of the control level (Fig. 5B)Citation . About 67% of the mitoses in BT549 overexpressing Syk were morphologically aberrant compared with 13.6% of the control vector cells (i.e., more than two spindle poles, Fig. 5ACitation , for examples).



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  		Fig. 5. Effects of Syk on mitosis and apoptosis. A and B, Visualization of mitotic cells by immunofluorescence staining of {alpha}-tubulin. BT549 cells stably transfected with either pCA1F or pCA1F-Syk as described in Fig. 3ACitation were grown for 6 days on coverslips, fixed in methanol, and processed for immunostaining (see Materials and Methods). Arrows point to mitotic spindles of dividing cells (A and B). Low magnification view of BT549 cells transfected with pCA1F-Syk and stained with antibodies against {alpha}-tubulin, at left. At right, example of a multipolar spindle at higher magnification (arrow). Scale bars = 10 µm. About 1,000 cells per coverslip were counted, and the population was divided between cells in mitosis (containing either bipolar or multipolar spindles, arrows) and not mitotic. These numbers are presented in B. C and D, DNA-damage induced apoptosis in BT549 breast carcinoma cells. BT549 cells stably transfected with pCA1F or with pCA1F-Syk as described in Fig. 3ACitation were untreated (UT) or exposed to 40 µmol/L etoposide for 72 hours (VP-16), and Annexin V binding activity was determined by FACSscan flow cytometry. Apoptotic cells are defined as Annexin V-positive/propidium iodide-negative cells. C, scatter plot of the results from flow cytometry. D, time course of apoptosis in response to etoposide. Cell death value is expressed as percentage of total cell number in the sample at the indicated times. Data presented are the mean values determined from triplicate experiments; bars, ±SE.

 
Apoptosis.
Previous studies show that BT549 breast carcinoma cells are responsive to various cell death stimuli including DNA damage (28) . Consequently, we next examined whether Syk is involved in mediating the susceptibility of BT549 cells to apoptosis induced by the DNA-damaging drug, etoposide, and ionizing radiation. We found that both BT549/pCA1F-Syk and BT549/pCA1F transfectants were capable of activating apoptosis in response to etoposide (Fig. 5, C and D)Citation or ionizing radiation (data not shown). However, we did not observe a significant difference in apoptotic response between Syk-expressing BT549 cells and vector-control cells (Fig. 5, C and D)Citation . Small differences in apoptosis may have been undetected, however, because Syk expression was gradually lost over the course of >2 weeks.

Src Activity.
Sustained Src activity is known to be associated with several misregulated cellular activities in cancer cells such as uncontrolled proliferation, high motility, and increased protease secretion and activity. We investigated whether the introduction of Syk into BT549 was affecting the activity of Src. Src activity is increased in BT549 due to the endogenous overexpression of epidermal growth factor receptors (29) . Stable populations of BT549 overexpressing pCA1F-Syk from independent transfection experiments were selected and grown in serum-containing medium. After 48 hours, cells were lysed, and total cell lysates were run on SDS-PAGE and blotted on nitrocellulose paper. Blots were probed with an antibody against the activated form of Src, Src-pY418 (Fig. 6ACitation , Src-pY418). Introduction of Syk resulted in a down-regulation of Src-Y418 tyrosine phosphorylation and, therefore, activity detected by Western blotting comparing intensity of anti-Src-pY418 to actin (Fig. 6A)Citation . To determine the level of Syk in transfected cells compared with cells that endogenously express Syk, lysates of MCF-7 breast cancer cells were compared with BT549 transfected with vector alone or pCAIF-Syk (Fig. 6B)Citation . Syk in stably transfected cells was expressed from ~1.2- to ~4-fold over the levels expressed in MCF-7 cells (Fig. 6B)Citation . Expression of Syk in MCF-7 cells is comparable with that in MCF-10A immortalized breast epithelial cells other Syk-positive breast cancer cells and K652 erythroleukemic cells (1) .

In addition, in a separate experiment, Src was immunoprecipitated from total cell lysates of cells transfected with pCA1F-Syk or the control plasmid, and its kinase activity was measured in an in vitro kinase assay using [{gamma}-32P]ATP and a synthetic peptide specific for Src as substrate (Fig. 6C)Citation . Once again, the kinase activity of Src, measured as picomoles of 32P incorporated in the substrate/minute, was lower in the Syk transfected cells. Down-regulation was ~30% of the total activity.

These experiments were repeated using enhanced green fluorescent protein-Syk and transiently transfected, nonselected cells. Western blots reveal that expression of enhanced green fluorescent protein-Syk (see Materials and Methods) did not influence the level of Src protein (Fig. 6DCitation , compare enhanced green fluorescent protein with enhanced green fluorescent protein-Syk). Enhanced green fluorescent protein-Syk was expressed at ~7.8-fold higher levels than endogenous Syk found in MCF-7 cells, when lysates were compared with equal protein loading (Fig. 6D)Citation . In this population, Src tyrosine kinase assay was suppressed to ~59% in enhanced green fluorescent protein-Syk-transfected BT549 cells (Fig. 6D)Citation . The level of Src tyrosine kinase activity in MCF-7 cells was 38% of that observed in pCA1F vector-transfected BT549 cells (Fig. 6D)Citation . Thus, the increased expression of Syk resulted in increased suppression of Src kinase activity compared with the previous experiment in which BT549 were transfected with pCA1F-Syk and antibiotic selected for 2 to 3 weeks.


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We have shown previously that Syk mRNA is commonly found in breast epithelial cells, whereas it is lost in invasive lesions (1) . Also, loss of Syk mRNA was reported associated with metastasis and poor prognosis and was found to be independent from other prognostic markers such as estrogen receptor and Her2 expression (2) , suggesting a novel role for Syk as a prognostic marker. In this report we extended the study of Syk expression to normal breast tissue from mammoplasty reduction, normal tissue adjacent to cancer lesions, hyperplasia, and DCIS, as well as invasive tissue, to determine whether the loss of Syk was common to the different types of cancer lesions or if it was a specific feature of invasive tissue. Of 113 cases studied, 18 were from mammoplasty reduction (no tumor lesions), 43 had only DCIS with or without hyperplasia, and 52 had also invasive components. Expression of Syk mRNA was studied by in situ hybridization. Specificity of the probe was confirmed by immunohistochemistry with a specific antibody against Syk. We found that the expression of Syk mRNA was progressively lost from normal to hyperplasia and DCIS to invasive tissue. Most importantly, Syk was already lost in normal tissue adjacent to cancer lesions compared with normal breast tissue in the absence of disease.

Yuan et al. (3) found recently that DNA methylation was responsible for loss of Syk in invasive tissues relative to adjacent normal tissue. Methylation of the Syk gene correlated with reduced Syk mRNA in invasive tissues and cells. More recent studies have additionally implicated methylation of Syk in the suppression of Syk mRNA and protein associated with T-lineage acute lymphoblastic leukemia cells (30) . However, our data suggest that other mechanisms for loss of Syk mRNA are operative, because in situ detection of Syk mRNA revealed a progressive loss of Syk beginning with normal adjacent tissue.

Although Syk showed a general trend to be progressively lost in breast cancer, in some cases the levels of Syk in invasive tissues were comparable with the level in mammoplasty reduction samples. This observation points again to the heterogeneous nature of cancer and to the need to discover new and more specific markers for the different subsets of cancer to develop more efficient anticancer drugs. The nature of Syk as a tumor and metastasis suppressor gene together with its uneven expression in cancer lesions makes it a very good subject for future studies of cancer biology in specific subsets of patients.

We have shown previously that malignant cells transfected with Syk and injected orthotopically into mice grew tumors at a slower rate and were characterized by a high number of multipolar spindles, suggesting that Syk might block tumor growth in vivo by inducing abnormal mitosis (1) . Here we confirmed in vitro that Syk is indeed responsible for increasing the number of aberrant mitoses as seen in the xenographed tumors. We also showed that Syk is able to suppress cell proliferation in vitro. Our in vitro model with BT549 cells transfected with Syk or the vector control showed that Syk-expressing cells had a proliferation rate of ~65% of the vector control. It is important to mention, however, that Syk was able to inhibit cell proliferation in vitro only in the presence of serum-containing medium. At lower concentrations of serum (0% and 1%), there was no difference between Syk-expressing BT549 and the vector control (data not shown). Interestingly, we were not able to detect a difference in proliferation of MDA-MB-435 cells cultured in vitro.6 MDA-MB-435 have been suggested recently to be melanoma cells rather than breast cancer cells (31 , 32) . Perhaps the difference in effects of Syk on proliferation is related to origin of tumor cells. Alternatively, differences might be related to the mechanism of enhanced proliferative abilities in tumor cells, such as overexpression of growth factor receptors including epidermal growth factor receptor.

Moreover, {alpha}-tubulin staining of BT549 grown on coverslips in the presence of serum-containing medium confirmed a lower mitotic index and a higher number of abnormal mitotic events for the cells expressing Syk. In the control cells, 13.5% of all of the mitoses were aberrant, whereas for the Syk-expressing cells the percentage of aberrant mitosis was >60%. It is not uncommon for cancer cell lines to show multipolar mitotic spindles (ref. 33 , for example) and lack mitotic spindle checkpoint(s). One proposed model of multipolar spindle formation is the splitting of pericentriolar material in the absence of cell division and centriole replication (34) . Syk is known to mediate tubulin dynamics (35) and to phosphorylate tubulin-binding proteins, such as VAV and Cbl (36) . Because the dynamic nature of microtubules is fundamental for spindle morphogenesis and chromosome movement during mitosis, the ability of Syk to modulate tubulin polymerization and/or binding and activity of microtubule-binding proteins in cells that have otherwise lost spindle checkpoints may explain the increased number of multipolar spindles seen in Syk-expressing BT549. A role for Syk in the inhibition of in vitro cell proliferation by modulation of tubulin dynamics is also supported by the finding that some tubulin-targeting drugs induce the formation of multipolar spindles and block mitosis, as well as cell proliferation (37) .

Syk activity is regulated by Src in hematopoietic cells (38) . However, a potential role for Syk in the regulation of Src has thus far not been investigated in breast epithelium cells. The observation that Syk-deficient cells such as MDA-MB-231 and BT549 have high Src activity (39) raised the question of whether Syk could negatively regulate Src. Indeed, we were able to demonstrate using two different approaches that, at least in vitro, reintroduction of Syk in Syk-negative Src-positive BT549 cells induced a partial inhibition of Src activity. The significant decrease in Src activity in cells transiently transfected with wild-type Syk can be interpreted in several ways. First, the trivial explanation is that Syk overexpression to high levels is associated with a strong suppression of Src activity. Indeed, soon after transfection, enhanced green fluorescent protein-Syk was found in 23% of the population, and Src activity was suppressed to ~59% of controls. In cells cultured for several weeks after transfection and selected for expression using G418, Syk expression was lower, and a less profound effect on Src activity was observed. If Src activity had been completely suppressed in the enhanced green fluorescent protein-Syk transfected cells, i.e., no activity in 23% of the cells in the enhanced green fluorescent protein-Syk transfection experiment, we would have expected a maximum 23% reduction in Src activity measured in the total cell population;, however, we observed a reduction of 41% (to ~59% of control). Similarly, in stably transfected pCAF1-Syk cells, ~6% of the cells were transfected, and we would have expected a maximum 6% reduction, yet we observed a ~30% reduction in Src activity in the total cell population. Therefore, to explain this apparent discrepancy, another possibility is that transfection with Syk induces a paracrine effect. The total cell population of transfected and nontransfected cells might be induced to down-regulate Src activity via a change in secretion of factor(s) by transfected cells. Studies are under way to examine Src activity in individual cells as a function of the level of Syk expression.

Src activity is associated with proliferating tumor tissue (40) . Syk negatively regulates Src activity in vitro, and the level of Syk mRNA was significantly associated with the proliferative activities of tumor tissues in vivo. This suggests a role for Syk in tumor suppression by regulation of cell proliferation through inhibition of Src activity. Because Src plays multiple and critical roles in cellular motility and signaling via the extracellular matrix, the tumor suppressor role of Syk might also be associated with these activities. For example, Syk might act as a negative regulator of cytoskeletal rearrangements involved in cell motility of breast epithelial cells. Indeed, loss of Syk is associated with increased invasiveness of breast cancer cells and increased cell migration (1 , 41) .7

It was reported recently that the alternatively spliced form of Syk, lacking 23 amino acids in the linker domain, is differentially detected in tumor cells in vitro and in some tumor specimens using Western blotting of cell or tissue lysates but was not detected in adjacent normal tissues (42) . In the present study, we detected mRNA of both the long and short form of Syk, because the probe was derived from bp 472 to 611 (1) , whereas the sequence that is deleted in the short form of Syk begins at bp 995 (43) . Because we were able to localize Syk expression in tissues, we eliminated the contribution of contamination via blood cells from our analyses. However, determination of the localization of the long and short, alternatively spliced form of Syk awaits utilization of specific probes for detection of Syk in tissue sections before contributions of each to breast cancer progression can be evaluated.

From our studies, we conclude that the overall level of total Syk progressively declines, and its early loss in normal adjacent tissues implies the existence of other mechanisms of loss in addition to hypermethylation. In vitro, Syk increased the number of mitotically aberrant cells and down-regulated Src activity.


   FOOTNOTES
 
Grant support: DAMD17–00-1–0273 and NIH R01DK48910 (S. C. Mueller). We also thank the Histopathology and Tissue, Microscopy and Imaging, Tissue Culture, Biostatistics, Research Computing, and Flow Cytometry Shared Resources of Lombardi Cancer Center, which are partially supported by NIH Grant 1P30-CA-51008 (Cancer Center Support Grant to Lombardi Cancer Center).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Requests for reprints: Susette C. Mueller, Department of Oncology, Georgetown University Medical Center, E301 Research Building, 3970 Reservoir Road NW, Washington, DC 20057-1469. Phone: 202-687-8484; Fax: 202-687-7505; E-mail: muellers{at}georgetown.edu

6 P. Coopman, M. Do, S. Mueller, unpublished observations. Back

7 M. Moroni and S. Mueller, unpublished observations. Back

Received 11/10/03. Revised 8/ 6/04. Accepted 8/16/04.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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