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Carcinocinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) is an intercellular adhesion molecule that is overexpressed in a wide variety of human cancers, including colon, breast, and lung and is associated with tumourigenesis, tumour cell adhesion, invasion, and metastasis. In the present study, we showed that CEACAM6 was overexpressed in a panel of oestrogen receptor (ERα)-positive human breast cancer cell lines (MCF-7:5C and MCF-7:2A) that have acquired resistance to oestrogen deprivation and this overexpression was associated with a more aggressive invasive phenotype in vitro. Expression array analysis revealed that MCF-7:5C and MCF-7:2A cells overexpressed CEACAM6 mRNA by 27-fold and 12-fold, respectively, and were 6 to 15-times more invasive compared to non-invasive wild-type MCF-7 cells which expressed low levels of CEACAM6. Suppression of CEACAM6 expression using small interfering RNA (siRNA) completely reversed migration and invasion of MCF-7:5C and MCF-7:2A cells and it significantly reduced E-cadherin, Akt, and expression in these cells. In conclusion, our findings establish CEACAM6 as a unique mediator of migration and invasion of drug resistant oestrogen deprived breast cancer cells and suggest that this protein could be an important biomarker of metastasis.
Carcinocinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) is a glycosylphosphatidylinositol-anchored cell surface protein that functions as a homotypic intercellular adhesion molecule (1). It is overexpressed in a number of human malignancies including pancreatic cancer, gastrointestinal cancer, and breast cancers (2, 3) and increased levels of CEACAM6 are inversely correlated to the differentiation state of cancer cells. Previous studies have shown that CEACAM6 is overexpressed in pancreatic adenocarcinoma cells and its overexpression is associated with greater in vivo metastatic ability and increased invasiveness and migration (4, 5). More recently, Poola and coworkers (6) reported that the expression of CEACAM6 in atypical ductal hyperplasia was associated with the development of invasive breast cancer (IBC). Currently, however, the role of CEACAM6 overexpression in breast cancer migration and invasion is not known.
Invasion and metastasis are the hallmarks of cancer malignancy and they are the primary cause of patient mortality during breast cancer progression (7). Invasion refers to the ability of cancer cells to penetrate through the membranes that separate them from healthy tissues and blood vessels and metastasis refers to the spreading of cancer cells to other parts of the body (8). In order for a transformed cell to metastasize, it must first lose adhesion, penetrate and invade the surrounding extracellular matrix (ECM), enter the vascular system, and adhere to distant organs (8). These processes require extensive alterations in gene expression profiles, including the down-regulation of genes involved in cell anchorage and the up-regulation of genes involved in cell motility and matrix degradation (7, 9, 10).
Aromatase inhibitors (AIs) are anti-oestrogen agents that suppress oestrogen production in peripheral tissues and breast tumours by inhibiting or inactivating aromatase, the enzyme which catalyses the conversion of androgens to oestrogens in postmenopausal women (11). Several randomized trials (12–15) have shown that third generation AIs are superior to adjuvant tamoxifen in terms of improved disease-free survival and less side effects. Unfortunately, one of the consequences of prolonged oestrogen deprivation/suppression is the development of drug resistance (16, 17). Previous studies have shown that acquisition of tamoxifen resistance in breast cancer cells is associated with a significant increase in motility and invasion (18, 19) along with increased CEACAM6 expression (20), however, it is unknown whether acquired resistance to oestrogen deprivation affects tumour cell migration and invasion and whether CEACAM6 plays a role in this process.
In the present study, we investigated the role of CEACAM6 in cellular migration and invasion of breast cancer cells that have acquired resistance to oestrogen deprivation. We found that CEACAM6 was significantly overexpressed in oestrogen-deprived MCF-7:5C and MCF-7:2A breast cancer cells and that these cells were markedly more migratory and invasive than parental MCF-7 cells. Suppression of CEACAM6 expression by small interfering RNA (siRNA) completely reversed the invasive phenotype of MCF-7:5C and MCF-7:2A cells. E-cadherin and β-catenin were also significantly reduced in these cells. The mechanism of action of CEACAM6 appears to involve, in part, the c-Src and Akt signaling pathways.
17 beta-oestradiol was purchased from Sigma Chemical Co. (St Louis, MO); PP2 was purchased from EMD Biosciences, Inc. (La Jolla, CA); LY294002 was purchased from Promega (Madison, WI); fulvestrant was obtained as a generous gift from AstraZeneca (Macclesfield, United Kingdom); Affymetrix Human Genome U133 Plus 2.0 Arrays were purchased from Affymetrix (Santa Clara, CA); fetal bovine serum (FBS), cell culture medium, and other reagents were purchased from Invitrogen (Carlsbad, CA).
Wild-type MCF-7 human breast cancer cells (21) were obtained from Dr. Dean Edwards (University of Texas, San Antonio, TX) and were maintained in fully ooestrogenized medium (RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), 2 mM glutamine, 100 U/mL penicillin, 100 µg/mL streptomycin, 1X non-essential amino acids, and bovine insulin at 6 ng/mL (Sigma-Aldrich, St. Louis, MO). MCF-7:5C (21–23) and MCF-7:2A (24) cells were clonally selected from parental MCF-7 cells following long-term culture (>1 year) in phenol red-free RPMI 1640 media containing 10% dextran-coated charcoal stripped FBS (SFS).
Total RNA was prepared using the Qiagen RNeasy Mini kit. A DNase I digestion step was included to eliminate DNA contamination. cRNA was generated, labeled, and hybridized to the Affymetrix Human Genome U133 Plus 2.0 Arrays by the Northwestern University Genomics Core (Chicago, IL). Arrays were washed, stained, and scanned according to the directions detailed in the Affymetrix GeneChip® Expression Analysis Technical Manual.
Assessment of data quality was conducted following default guidelines in the Affymetrixs GeneChip® Expression Analysis Data Analysis Fundamentals Training Manual. Data was extracted and normalized using Affymetrix Microarray Suite (MAS5.0) following recommended protocols for background and chip-correction. Global scaling for average signal intensity for all arrays was set to 500. Four biological replicates from each of the three cell lines were arrayed to determine consistent and reproducible patterns of gene expression. All but one array showed a high degree of reproducibility within a set of replicate hybridizations, leaving at least three array replicates per cell line for further analysis. Genes across all arrays with an expression intensity <70 were removed. To eliminate genes with variable expression within a group of replicates, normalized gene intensity ratios (signal intensities divided by the median gene intensity all hybridizations) were derived, then the standard deviation of the log-transformed normalized intensity ratios were calculated for each group of replicates. Genes with a standard deviation >0.15 were excluded. Lastly, to filter for genes with variable expression between cell lines, genes were retained that showed a standard deviation of >0.3. A total of 904 genes met the filtering criteria described and were examined by hierarchical clustering using resources available at TGen1. Uncentered Pearson’s correlation with average linkage was used on log2-transformed data, with induced genes indicated in red and repressed genes in green. Random permutation analysis was performed as previously described (25) using 10,000 permutations. Genes with a p-value <0.01 and an alpha value <0.01 were used for gene ontology analysis.
Cell proliferation assay was performed as previously described (22). The DNA content of the cells was determined using a Fluorescent DNA Quantitation kit (Bio-Rad Laboratories, Hercules, CA). For each analysis, three replicate wells were used, and at least three independent experiments were performed.
Western blot analyses were performed as previously described (22). Separated proteins were transferred onto nitrocellulose membranes (Milllipore) and incubated overnight at 4°C with the respective primary antibodies; CEACAM6 and CEACAM5 (Signet Laboratories, Dedham, MA); ERα N-cadherin, β-catenin, CXCR4, MMP9, E-cadherin, and CD44 (Santa Cruz Biotechnology, Santa Cruz, CA); fibronectin (Chemicon International, Temecula, CA); c-Src and p-SrcTyr529 (Biosource International, Carmarillo, CA); AKT and p-AKTSer473 (Cell Signaling Technology, Beverly, MA); and β-actin (Sigma Chemical Co, St Louis, MO). Secondary antibodies conjugated to horseradish peroxidase (Santa Cruz Biotechnology) were used with an enhanced chemiluminescence (ECL) kit (Amersham, Arlington Heights, IL) to visualize the resolved proteins.
Total RNA was extracted using the RNeasy mini kit (Qiagen, Valencia, CA) according to the manufacturer's instructions. Ten micrograms of total RNA for each sample was converted to first-strand cDNA using SuperScript III with a combination of random hexamers and oligo(dT) as primers (Invitrogen). Quantitative real-time PCR assays were done as previously described (22) with the Taqman Universal or SYBR Green PCR Master Mixes and an ABI 7700 sequence detection system (PE Applied Biosystems, Foster City, CA). The ERa forward and reverse primers were 5’-AAGAGGGTGCCAGGCTTTGT-3’, and 5’-CAGGATCTCTAGCCAGGCAC AT-3’, respectively. The ERα probe was 5’-[FAM]-ATTTGACCCTCCATGATCAGGTCC ACC-[TAMRA]-3’. The forward and reverse primers for CEACAM6 were synthesized by Sigma Genosys (Sigma-Aldrich). The sequences for CEACAM6 forward and reverse primers were 5’-GACGTTTGTGTGGATTGCTGGAACGC-3’ and 5’-TGCCACGCAGCCTCTAACC-3’. The reporter dye at the 5'-end of each probe was FAM and the quencher dye at the 3'-end was TAMRA. The 18S ribosomal RNA (18S rRNA) gene was used as an endogenous control to normalise for differences in the amount of total RNA in each sample, 18S rRNA primers and probes were purchased from Applied Biosystems. Relative expression of the target gene was calculated using the 2 delta CT method described previously (26). (Relative expression = 2−ΔCT; where ΔCT = CT (Target gene) − CT (endogenous control gene)) where 18S rRNA is the endogenous control gene. To determine relative RNA levels within the samples, standard curves for the PCR were prepared by using cDNA from one sample and making twofold serial dilutions covering the range equivalent to 20–0.625 ng RNA (for 18S rRNA analyses, the range was 4–0.125 ng).
Cell migration was measured in a Boyden chamber using Transwell filters obtained from Corning (Cambridge, MA). Cells (1 × 105) in 0.5 mL serum-free medium were placed in the upper chamber, and the lower chamber was loaded with 0.8 mL medium containing 10% SFS. Cells that migrated to the lower surface of filters were stained with Wright Giemsa solution, and five fields of each well were counted after 24 or 48 hours of incubation at 37°C with 5% CO2. Three wells were examined for each condition and cell type, and the experiments were repeated in triplicate. Cell invasion assay was performed using the Chemicon cell invasion kit (Chemicon International, Temecula, CA) in accordance with the manufacturer's protocol. Cells (1 × 105/ml) were seeded onto 12-well cell culture chamber using inserts with 8 µM pore size polycarbonate membrane over a thin layer of extracellular matrix. Following incubation of the plates for 48 h at 37°C, cells that had invaded through the ECM layer and migrated to the lower surface of the membrane were stained and counted under the microscope in at least ten different fields and photographed.
CEACAM6-specific siRNA (Silencer™ Predesigned siRNA; sense: GCCCUGGUGUAUUU UCAUtt, antisense: AUCGAAAAUACAC CAGGGCtg) (AM16704) and scramble sequence control siRNA (Silencer™ Negative Control siRNA) were purchased from Ambion (Austin, TX). Transfection complexes were prepared in Opti-MEM serum-free medium (Invitrogen) by mixing 0.3 µL of siPORT NeoFX transfection reagent (Ambion) and 10 nM CEACAM6 siRNA or negative control siRNA (Ambion). Cells (9×104 cells per well) were reverse-transfected in 12-well plates simultaneously with addition of transfection complexes. The medium was replaced with phenol red-free RPMI supplemented with 10% SFS 24 h after transfection and cultures were harvested for CEACAM6 protein and mRNA analyses.
Statistical analyses were performed using Microsoft Excel (Seattle, WA). Differences between groups were evaluated using Student's t test. Data were considered significant if p < 0.05.
The growth of oestrogen deprived MCF-7:5C and MCF-7:2A cells is compared to parental MCF-7 cells in Fig. 1A. Both MCF-7:5C and MCF-7:2A cells grew robustly in the absence of oestrogen whereas MCF-7 cells grew minimally without oestrogen. The doubling times were 2.7, 3.4, and 6 days for MCF-7:5C, MCF-7:2A, and MCF-7 cells, respectively. We also examined cell morphology changes associated with resistance to long term oestrogen deprivation using phase-contrast microscopy. Fig. 1B shows that MCF-7 cells grew as a uniform monolayer of tightly associated cells with limited cell spreading but distinct cellular boundaries whereas oestrogen deprived MCF-7:5C and MCF-7:2A cells grew in a less uniform monolayer with cellular boundaries that were obscured. ERα mRNA and protein expression were also significantly increased in MCF-7:5C and MCF-7:2A cells compared to MCF-7 cells and treatment with oestradiol or the pure antioestrogen fulvestrant significantly down regulated its expression (Fig. 1C and D) in all three cell lines. Overall, these results show that oestrogen deprivation increases ERα expression and alters the morphology of MCF-7:5C and MCF-7:2A cells.
Transcriptional profiling of parental MCF-7 cells and oestrogen deprived MCF-7:5C and MCF-7:2A cells was performed using Affymetrix Human Genome U133 Plus 2.0 Array. Two-dimensional hierarchical clustering was performed to analyze differences in gene expression patterns between MCF-7 cells and MCF-7:5C and MCF-7:2A cells. Data filtering identified 904 genes that were significantly altered between MCF-7:5C and MCF-7:2A cells and parental MCF-7 cells (Fig. 2A and Supplemental Fig. S1). The sample dendogram showed that MCF-7:2A cells and MCF-7 cells clustered more closely whereas MCF-7:5C cells clustered on a more distant branch, suggesting that MCF-7:2A cells are more similar to parental MCF-7 cells than MCF-7:5C cells (Fig. 2A). In order to define cell signalling mechanisms that differed significantly between parental MCF-7 and MCF-7:5C and MCF-7:2A cells, random permutation weighted gene analysis was performed as described in the methods. A comparsion of MCF-7 expression data with that of MCF-7:5C and MCF-7:2A revealed that 4068 genes were highly differentially expressed (Supplemental Table 1). Gene Ontology analysis showed a significant number of genes associated with cell cycle control, proliferation, growth factor signalling, cell adhesion, and motility and invasion. In particular, we found that CEACAM6 was overexpressed by 27-fold in MCF-7:5C cells and 12-fold in MCF-7:2A cells (Fig. 2B) and it was highly weighted in our random permutation analysis (p-value <.0001) (Supplemental Table 1).
To confirm our microarray data, CEACAM6 mRNA expression was determined by quantitative RT-PCR. Fig. 3A shows that CEACAM6 mRNA was significantly upregulated in oestrogen-deprived MCF-7:5C and MCF-7:2A cells compared with parental MCF-7 cells. Similarly, by Western blotting, CEACAM6 protein was undetectable in MCF-7 cells but was strongly expressed in MCF-7:5C and MCF-7:2A cells (Fig. 3B). Other invasion proteins such as CEACAM5, MMP-9, CXCR4 and CD44 were also markedly elevated in MCF-7:5C and MCF-7:2A cells compared to MCF-7 cells (Fig. 3B). This finding is consistent with a recent study by Mackay and coworkers (27) which revealed that many genes associated with extracellular matrix remodeling were significantly upregulated following aromatase inhibitor treatment of primary breast tumours.
Since MCF-7:5C and MCF-7:2A cells overexpressed several invasion genes, we next assessed the migratory and invasive potential of these cells in vitro. Cell migration was measured using a modified Boyden chamber assay with 10% SFS as a chemoattractant. As shown in Fig. 3C, MCF-7:5C and MCF-7:2A cells had the highest numbers of migrating cells compared to MCF-7 cells; a phenotype that correlated with CEACAM6 expression. Similar results were obtained when the different cell lines were tested for their ability to invade through membranes coated with Matrigel. Fig. 3D shows that MCF-7:5C and MCF-7:2A cells had the highest number of invading cells while MCF-7 cells were noninvasive. The invasive ability of the cell lines was as follows: MCF-7:5C > MCF-7:2A > MCF-7.
To test the hypothesis that CEACAM6 is required for cell migration and invasion, we used siRNA to suppress CEACAM6 expression. MCF-7:5C cells were transfected with CEACAM6-specific or control (scrambled sequence) siRNA and Western blot analysis was performed 72 hours post transfection. Fig. 4A (top) shows that CEACAM6 protein was significantly suppressed (75–85%) in MCF-7:5C cells transfected with the CEACAM6-specific siRNA but not the control siRNA. siRNA suppression of CEACAM6 expression was also confirmed at the transcript level using qRT-PCR at 48 hours following transfection (Fig. 4A bottom). To clarify the role of CEACAM6 in cell invasion, MCF-7:5C cells were pretreated with CEACAM6 siRNA or control siRNA for 48 hours and invasion was measured over the subsequent 48 hours. Fig. 4B shows that CEACAM6 siRNA almost completely reversed the invasiveness of MCF-7:5C cells whereas control siRNA did not affect cell invasion. The invasiveness of MCF-7:5C cells was inhibited by nearly 80% when CEACAM6 expression was suppressed. A similar trend was observed for cell migration (data not shown). Suppression of CEACAM6 also significantly reduced phosphorylated Akt and phosphorylated c-Src in MCF-7:5C cells (Fig. 4C). E-cadherin and β-catenin were also significantly reduced in MCF-7:5C and MCF-7:2A cells whereas pAkt and N-cadherin were significantly upregulated in these cells compared to parental MCF-7 cells (Fig. 4D). Similar experiments performed in MCF-7:2A cells also showed a dramatic reduction (60%) in invasion following CEACAM6 suppression (data not shown).
We also examined whether CEACAM6 expression is hormonally regulated in MCF-7:5C and MCF-7:2A cells. As shown in Fig. 5A and B, oestradiol completely down-regulated CEACAM6 mRNA and protein expression in MCF-7:5C and MCF-7:2A cells. This downregulation was an ERα-mediated event since pretreatment with the antioestrogen fulvestrant, which is known to degrade ERα (28, 29), was able to reverse the inhibitory effect of oestradiol on CEACAM6 protein in both cell lines (Fig. 5B). Fulvestrant also completely counteracted the anti-invasive effects of oestradiol in MCF-7:5C cells (Fig. 5C). Interestingly, oestradiol enhanced the invasiveness of parental MCF-7 cells (Fig. 5D) without significantly changing CEACAM6 protein level in these cells (Fig. 5B).
Previous studies have reported that CEACAM6 cross-linking initiates c-Src-dependent cross-talk between CEACAM6 and αvβ3 integrin, leading to increased ECM-adhesion and invasion (30). We therefore determined c-Src kinase activity in oestrogen-deprived MCF-7:2A and MCF-7:5C cells by measuring phosphorylation of c-Src at Tyr529. Both MCF-7:5C and MCF-7:2A cells showed significantly elevated levels of phosphorylated c-SrcY529 compared to parental MCF-7 cells and treatment with the c-Src kinase inhibitor PP2 significantly reduced the invasiveness of MCF-7:5C and MCF-7:2A cells (Supplemental Fig. S2). Inhibition of Akt phosphorylation using the PI3K inhibitor LY294002 also significantly reduced cell growth and invasion of these cells (Supplemental Fig. S2), thus suggesting an important role for the c-Src and Akt signaling pathways in invasion.
Despite advances in detection and treatment of metastatic breast cancer, mortality from this disease remains high because current therapies are limited by the emergence of therapy-resistant cancer cells. In the present study, we showed that oestrogen deprivation significantly increased the motility and invasiveness of two ERα-positive human breast cancer cell lines that have acquired resistance to oestrogen deprivation and that these cells overexpressed the invasive gene CEACAM6. Furthermore, knockdown of CEACAM6 expression completely inhibited the invasiveness of MCF-7:5C and MCF-7:2A cells and caused a reduction in phosphorylated c-Src and pAkt expression. A significant reduction in E-cadherin and β-catenin was also observed in MCF-7:5C and MCF-7:2A cells compared to parental MCF-7 cells. To our knowledge, this study is the first to demonstrate a critical role for CEACAM6 in migration and invasion of breast cancer cells that have acquired resistance to oestrogen deprivation.
Previous studies have reported that overexpression of CEACAM6 in pancreatic adenocarcinoma cells is associated with enhanced cellular invasiveness and increased metastatic potential in vivo and that this effect is completely attenuated by suppression of CEACAM6 expression (4). Recently, Scott and coworkers (20) reported that CEACAM6 was upregulated by 20-fold in tamoxifen-resistant MCF-7 cells compared to tamoxifen-sensitive cells and that hormone sensitivity could be partially restored in the tamoxifen-resistant cells by siRNA silencing of CEACAM6. This in vitro data was substantiated in clinical breast cancer where it was demonstrated that CEACAM6 was overexpressed in primary breast tumours that subsequently relapsed following adjuvant tamoxifen and in a multivariate analysis, only CEACAM6 remained a significant predictor of recurrence (31). These findings are consistent with our present study which shows that CEACAM6 is significantly upregulated in oestrogen deprived breast cancer cells that have acquired resistance to oestrogen suppression and knockdown of CEACAM6 expression reverses the invasive phenotype of these cells. The fact that CEACAM6 is identified independently in two model systems using endocrine agents with distinct modes of action suggests that it may play an important role in endocrine resistance. Currently, the mechanism by which CEACAM6 facilitates invasion is not fully understood. However, there is evidence that CEACAM6, along with other GPI-anchored proteins, are capable of modulating the activity of intracellular tyrosine kinases such as c-Src (32, 33). In particular, studies by Duxbury and coworkers (30, 34) showed that c-Src activity was increased in CEACAM6-overexpressing BxPC3 human pancreatic cancer cells and decreased following suppression of CEACAM6 expression and that inhibition of c-Src activity significantly suppressed CEACAM6-mediated cellular invasiveness. We found that phosphorylated c-Src was significantly elevated in MCF-7:5C and MCF-7:2A cells and that suppression of CEACAM6 expression reduced its level in these cells. Pharmacological blockade of c-Src using the Src tyrosine kinase inhibitor pyrazolopyrimidine (PP2) also inhibited the invasiveness of MCF-7:5C and MCF-7:2A cells. In addition, we found markedly elevated levels of phosphorylated Aktser473 in MCF-7:5C and MCF-7:2A cells which were dramatically reduced following CEACAM6 suppression. Akt is a serine/threonine protein kinase that mediates cell survival, proliferation (35, 36), tumour cell migration and invasion and metastasis (37) and previous studies have shown that c-Src activates the PI3K/Akt signaling pathway (38). Thus, it is possible that activation of both c-Src and Akt might play a role in mediating CEACAM6-induced migration and invasion.
The epithelial-to-mesenchymal transition (EMT) plays a key role in metastasis and is characterized by the conversion of epithelial cancer cells to a more motile phenotype that facilitates invasion. A critical molecular feature of EMT is the downregulation of E-cadherin (39), a cell adhesion molecule present in the plasma membrane of most normal epithelial cells. E-cadherin acts de facto as a tumour suppressor inhibiting invasion and metastasis and is frequently repressed or degraded during transformation. In our study, E-cadherin and β-catenin were significantly decreased whereas N-cadherin was markedly increased in invasive MCF-7:5C and MCF-7:2A cells compared to noninvasive MCF-7 cells. In addition, our cell morphology studies showed EMT-like changes in MCF-7:5C and MCF-7:2A cells compared to MCF-7 cells. A variety of signal transduction pathways impinge on the regulation of E-cadherin levels or subcellular distribution. In particular, Akt/PKB has been shown to repress transcription of the E-cadherin gene which leads to conversion of epithelial cells into invasive mesenchymal cells (40). We have found that MCF-7:5C and MCF-7:2A cells both overexpress phosphorylated Akt and gene ontology analysis of expression data obtained for MCF-7:5C and MCF-7:2A cells reveal that the P13K/Akt signalling pathway is significantly (p = 0.002) altered compared to parental MCF-7 cells.
In conclusion, we have identified CEACAM6 as a critical gene in the regulation of migration and invasion of breast cancer cells that have acquired resistance to oestrogen deprivation. Since aromatase inhibitors are now considered the standard of care for the hormonal treatment of early breast cancer in postmenopausal women, this finding has important clinical implications for these patients because it suggests that extended use of aromatase inhibitors may potentially lead to the development of metastatic disease. CEACAM6 can thus serve as a powerful predictor of future recurrence and may also represent a promising new therapeutic target for breast cancer.
Effect of the Src inhibitor PP2 on Src phosphorylation and invasion of breast cancer cells. (A) MCF-7 cells, MCF-7:5C, and MCF-7:2A were treated with 10 µM PP2 for 24 h and Western blot analysis was performed on lysates for detection of total c-Src and c-Src-Tyr529. Anti-β-actin antibody was used as loading control. The relative ratio of c-Src-Tyr529 was calculated by densitometry (bottom). The bar graph (bottom) depicts the averages of the data obtained from three individual experiments, and data are expressed as means ± S.E. (B) The effect of c-Src inhibition on cellular invasion. Cells were treated for 48 hours. (C) Cell growth and cell invasion were determined as previously described. Cells were treated wit 10 µM PI3K inhibitor LY294002 for 7 days (top panel) or 48 hours (bottom panel). The experiments were repeated thrice in triplicates. Error bars indicate SE. *p<.001 (top panel) ; *p<.01 (bottom panel).
We thank Dr Chris Wambi (Department of Radiation Oncology, University of Pennsylvania, Philadelphia, PA) for his valuable comments and critical review of this manuscript. This work was supported by the NIH Career Development Grant 1K01CA120051-01A2; the American Cancer Society Grant IRG-9202714; the Department of Defense Breast Program under award number BC050277 Center of Excellence; Fox Chase Cancer Center Core Grant NIH P30 CA006927; Weg Fund of Fox Chase Cancer Center; and the Hollenbach Family Fund. The views and opinions of the author(s) do not reflect those of the US Army or the Department of Defense.
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The microarray results discussed in this publication have been deposited in NCBIs Gene Expression Omnibus (GEO, http://www.ncbi.nlm.nih.gov/geo/) and are accessible through GEO Series accession number GSE10879.
1Internet address: http://biodiscovery.tgen.org/microarray/