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Originally published In Press as doi:10.1074/jbc.M104209200 on July 9, 2001

J. Biol. Chem., Vol. 276, Issue 36, 33711-33720, September 7, 2001
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Functional Analysis of Csk and CHK Kinases in Breast Cancer Cells*

Cécile BougeretDagger , Shuxian JiangDagger , Iafa Keydar§, and Hava AvrahamDagger

From the Dagger  Division of Experimental Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02115 and the § Department of Cell Research and Immunology, Tel Aviv University, Ramat Aviv 69978, Israel

Received for publication, May 9, 2001, and in revised form, July 3, 2001

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In this report, we analyzed the expression and kinase activities of Csk and CHK kinases in normal breast tissues and breast tumors and their involvement in HRG-mediated signaling in breast cancer cells. Csk expression and kinase activity were abundant in normal human breast tissues, breast carcinomas, and breast cancer cell lines, whereas CHK expression was negative in normal breast tissues and low in some breast tumors and in the MCF-7 breast cancer cell line. CHK kinase activity was not detected in human breast carcinoma tissues (12 of 12) or in the MCF-7 breast cancer cell line (due to the low level of CHK protein expression), but was significantly induced upon heregulin (HRG) stimulation. We have previously shown that CHK associates with the ErbB-2/neu receptor upon HRG stimulation via its SH2 domain and that it down-regulates the ErbB-2/neu-activated Src kinases. Our new findings demonstrate that Csk has no effect on ErbB-2/neu-activated Src kinases upon HRG treatment and that its kinase activity is not modulated by HRG. CHK significantly inhibited in vitro cell growth, transformation, and invasion induced upon HRG stimulation. In addition, tumor growth of wt CHK-transfected MCF-7 cells was significantly inhibited in nude mice. Furthermore, CHK down-regulated c-Src and Lyn protein expression and kinase activity, and the entry into mitosis was delayed in the wt CHK-transfected MCF-7 cells upon HRG treatment. These results indicate that CHK, but not Csk, is involved in HRG-mediated signaling pathways, down-regulates ErbB-2/neu-activated Src kinases, and inhibits invasion and transformation of breast cancer cells upon HRG stimulation. These findings strongly suggest that CHK is a novel negative growth regulator of HRG-mediated ErbB-2/neu and Src family kinase signaling pathways in breast cancer cells.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Breast cancer is the leading cause of death in American women 30 to 70 years of age (1). The majority of breast carcinomas appear to be sporadic and become increasingly aggressive due to the acquisition of several successive, distinct genetic changes (2). In many cases, random onset of human breast cancer has been correlated with increased expression of the growth factor receptor ErbB-2/neu (also known as HER-2) (3), and with the increased activity of the Src family of non-receptor protein-tyrosine kinases (4). The overexpression of ErbB-2/neu is directly involved in mammary tumorigenesis and correlates with a poor prognosis in breast cancer (3). ErbB-2/neu is a member of the epidermal growth factor (EGF)1 receptor tyrosine kinase family (5). To date, no ligands that directly bind to the ErbB-2/neu receptor have been clearly identified. However, heterodimerization of ErbB-2/neu with other members of the EGF receptor family, the EGF receptor (or c-ErbB-1) and ErbB-3 (or c-HER-3), confers high affinity binding sites for EGF and heregulin (HRG) (also known as neu differentiation factor), respectively (6, 7). When the ErbB-2/neu receptor is activated, it undergoes autophosphorylation on five tyrosine residues located on its non-catalytic carboxyl terminus. The autophosphorylated tyrosine residues function as docking sites for proteins, such as Shc (8), phospholipase Cgamma (9), Ras-GTPase-activating protein (9), phosphatidylinositol 3'-kinase (10), and c-Src (11), that contain SH2 domains and also play a role in signal transduction pathways. Activation of Src family kinases in response to growth factor stimulation constitutes an essential step in the initiation of the mitogenic signal generated by several receptor tyrosine kinases (12). Src family kinases are activated in ErbB-2/neu-induced mammary tumors (4), and this elevated activity correlates with their capacity to physically associate with the ErbB-2/neu receptor (11).

Src family kinase activity is inhibited by the phosphorylation of a conserved, carboxyl-terminal tyrosine (13). The protein-tyrosine kinase responsible for this phosphorylation is Csk, Carboxyl-terminal src kinase (14, 15). Recently, several groups identified a second member of the Csk family, previously termed Matk (16), Ctk (17), Hyl (18), Ntk (19), Lsk (20), and Batk (21), and presently known as CHK for Csk Homologous Kinase (22). CHK and Csk are structurally related genes and share 53% amino acid identity overall and 59% amino acid identity within the catalytic domain (16-21). Like Csk, CHK contains Src homology (SH) domains 2 and 3, as well as the Src family catalytic domain (SH1), and lacks an amino-terminal myristoylation signal residue and is therefore localized to the cytoplasm. Additionally, like Csk, CHK phosphorylates in vitro the inhibitory carboxyl-terminal tyrosine of several Src family kinases, including c-Src (23-25), Lck (17, 19), Fyn (23), and Lyn (25-27). Unlike Csk, which is ubiquitously expressed, the expression of CHK is limited to neuronal and hematopoietic cells (16-21). Furthermore, CHK expression, unlike the constitutive expression of Csk, is induced by IL-2 in natural killer cells (20), by phytohemagglutinin in T lymphocytes (20), by the stem cell factor in human megakaryocytes (22), and by IL-4 and IL-13 in human monocytes (28).

Little is known about the role(s) of CHK in cellular physiology. It has been shown that the SH2 domain of CHK binds to several tyrosine-phosphorylated proteins that are involved in cell proliferation and differentiation. These proteins include the activated protein-tyrosine kinase receptor c-Kit in megakaryocytes (29, 30), the activated protein-tyrosine kinase receptor TrkA in PC12 cells (31), and the phosphorylated cytoskeletal protein paxillin in human blastic T cells (32). Recently, we reported that CHK expression was observed in primary human breast cancer specimens (70 of 80) by immunofluorescence staining but was not detected in normal human breast tissues (none of 19). Confocal microscopy analysis revealed co-localization of CHK with the ErbB-2/neu receptor in these primary breast cancer specimens (6 of 6) (33, 34). Furthermore, upon HRG stimulation of breast cancer cells, CHK associated with the HER-2/neu receptor (33). This association was receptor-specific (ErbB-2/neu) and ligand-specific (HRG), because no association was detected with the ErbB-2/EGF-receptor heterodimer upon EGF stimulation. CHK bound directly, via its SH2 domain, to the Tyr1253 autophosphorylation site of ErbB-2/neu (33, 34). The autophosphorylated tyrosine residues Tyr1253 of rodent neu and Tyr1248 of the human homologue ErbB-2 have been reported to be the most critical residues for the oncogenicity and transforming potential of ErbB-2/neu (35). Moreover, CHK was able to down-regulate ErbB-2/neu-activated c-Src kinase (34).

In this study, we analyzed the expression, kinase activities, and functions of Csk and CHK in primary human breast tumors, normal breast tissues, and breast cancer cell lines. Csk was highly expressed and kinase-active in both primary human breast cancer tissues and normal breast tissues, but its expression and kinase activity were not modulated by HRG. In contrast, CHK expression was observed in only some breast cancer tissues at low levels and was not detected in normal breast tissues. Moreover, CHK kinase activity could not be detected in these samples (due to the low level of CHK protein expression). However, upon stimulation with HRG, CHK kinase activity was significantly induced.

We further studied CHK function in HRG-mediated signaling and evaluated the anti-tumoral potential of overexpressing wild-type (wt) CHK protein in MCF-7 cells by stable transfection. We showed that overexpression of wt CHK significantly inhibited in vitro cell growth, transformation, and invasion induced upon HRG stimulation of MCF-7 cells. Tumor growth of wt CHK-transfected MCF-7 cells in nude mice was also significantly inhibited. In addition, in vitro c-Src and Lyn protein expression and kinase activities were down-regulated, and entry into mitosis was delayed in wt CHK-transfected MCF-7 cells. Taken together, these data indicate that CHK, but not Csk, is involved in HRG-mediated signaling and is potentially a novel negative growth regulator of this signaling pathway in breast cancer cells.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cell Lines and Tissue Samples-- Human megakaryocytic cells (Dami, MEG-01), normal breast cells (MCF-10A, HBL-100), and breast cancer cells (MCF-7 and ZR-75-1) were obtained from ATCC (American Type Culture Collection, Rockville, MD). Primary human breast tissues were obtained from the Cooperative Human Tissue Network (Eastern Division, Philadelphia, PA). Prior to stimulation with HRG, cells were starved overnight in serum-free medium and for an additional 4 h in fresh serum-free medium, then were induced with 10 nM HRG for 10 min. Recombinant human HRG (gamma HRG-beta 1, 177-244) was generously provided by Dr. Mark X. Sliwkowski (Genentech Inc., San Francisco, CA) (36).

DNA Amplification and Sequencing-- Total RNA from primary human breast cancer tissues and human breast cell lines was prepared by a standard protocol of lysis in guanidium isothiocyanate, followed by cesium chloride gradient centrifugation (37). Poly(A+) RNA was isolated, and CHK sequences were amplified by RT-PCR with degenerate oligonucleotide primers, as described previously (16). The PCR products of the amplified CHK were purified from the agarose gel, ligated into pUC19, and transformed into Escherichia coli DH5alpha , as described previously (16). Sequencing was carried out by the dideoxy chain termination method using a Sequenase kit (U.S. Biochemical Corp.).

Southern Blot Analysis-- PCR products prepared as described above were electrophoresed on a 2% agarose gel, denatured, neutralized, transferred to filters, and vacuum-blotted. The filters were baked at 80 °C for 2 h and then prehybridized according to the manufacturer's instructions. The probes used were the full-length CHK and Csk cDNAs, which were labeled by random priming as described previously (16). Hybridization was carried out as described previously (37) at 42 °C in buffer containing 50% (v/v) formamide. The blotted membrane was washed (37) at 62 °C and then subjected to autoradiography.

Western Blot Analysis-- For Western blot analyses, cells were scraped off the plates and lysed in cell lysis buffer (10 mM Tris-HCl, pH 7.4, 1 mM EDTA, pH 8.0, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 10% glycerol) containing 10 µg/ml aprotinin, 10 µg/ml leupeptin, 10 µg/ml pepstatin, 1 mM phenylmethylsulfonyl fluoride, and 1 mM sodium orthovanadate inhibitor for 45 min at 4 °C. Protein concentration was determined using a protein assay (Bio-Rad). 50 µg of total protein extracts were electrophoretically separated on 10% polyacrylamide-SDS gels, transferred to polyvinylidene difluoride membrane, and probed with antibodies against CHK (Lsk, Santa Cruz Biotechnology), Csk (Santa Cruz Biotechnology), Src (clone GD11, Upstate Biotechnology Inc.), Lyn (Santa Cruz Biotechnology), and actin (clone-4, Roche Molecular Biochemicals). Immunodetection was performed using the enhanced chemiluminescence system (ECL, Amersham Pharmacia Biotech).

Expression Vectors and Stable Transfection-- Wild-type human CHK cDNA (1.6 kilobase pairs) was cloned into the pcDNA3-neo mammalian expression vector with the FLAG epitope introduced to the 5'-end of the open reading frame of the CHK cDNA as previously described (33). The lysine (AAG) to arginine (GCG) mutation at position 262 in the ATP-binding site of the kinase domain of CHK (dead-kinase) was generated by site-directed mutagenesis using the QuikChange kit from Stratagene, with the sense primer 5'-GGGCAAAAGGTGGCCGTGGCGAATATCAAGTGTGATGTG-3' and the antisense primer 5'-CACATCACACTTGATATTCGCCACGGCCACCTTTTGCCC-3'. Transfection of MCF-7 cells was performed using LipofectAMINE (Life Technologies, Inc.) according to the manufacturer's protocol. The transfected cells were selected in 0.5 mg/ml Geneticin (G418, Life Technologies, Inc.). Positive transfectants were chosen based on their immunoreactivity on Western blots probed with polyclonal anti-CHK (Lsk, Santa Cruz Biotechnology, Santa Cruz, CA) and monoclonal anti-FLAG (M2, Sigma) antibodies.

Cell Growth Assay-- MCF-7 cells (105 cells/well) were spread in 24-well plates and starved in serum-free medium. Cells were then grown in serum-free medium alone or supplemented with 10 nM HRG. Viable cells were stained with 0.1% crystal violet. Staining was recovered with 2% deoxycholate and quantitated by spectrophotometry (490 nm).

Colony Forming Assay-- Transformation of cells was assessed by their ability to demonstrate anchorage-independent growth (38). MCF-7 cells (1.5 × 105 cells/well in 6-well dishes) were seeded in medium containing 0.3% agar (Sigma) and allowed to grow for 2 weeks before counting viable colonies (3 cells or more per colony).

Cell Invasion Assay-- The Matrigel invasion assay was performed as previously described (39) using 6.5-mm Transwell chambers (8-µm pore size, Costar). Matrigel was diluted in cold distilled water (2 µg/ml), added to the upper wells of the Transwell chambers, and dried in a sterile hood. The Matrigel was reconstituted with medium for 1 h at 37 °C before the addition of cells. Cells were starved in serum-free medium, then resuspended at a concentration of 1-2 × 106 cells/ml in serum-free medium containing 0.1% bovine serum albumin, alone or supplemented with 10 nM HRG. 100 µl of the cell suspension was added to each well, and conditioned NIH 3T3 medium (600 µl) was added to the bottom wells of the chambers. After 18 h, the cells that had not invaded were removed from the upper surface of the filters using cotton swabs. The cells that had invaded to the lower surface of the filters were fixed with methanol, stained with 0.2% crystal violet, and counted.

Tumor Growth in Nude Mice-- MCF-7 tumors were induced in 7- to 8-week-old female athymic nu/nu Swiss mice on day 0 by subcutaneous injection of 107 cells into the mammary fat pad (n = 6 animals for each group). Tumors were measured in three orthogonal dimensions every 5 days, and volumes were estimated assuming an ellipsoid shape as follows: (a × b × c)/2. Differences between tumors were compared using the two-tailed Mann-Whitney non-parametric rank test (two-sided). For Western blot analysis, mice were sacrificed at the end of the experiment (day 60) by cervical dislocation. Tumors were taken after skin incision using scissors and forceps, and total protein extracts were prepared and analyzed as described above (see "Western Blot Analysis").

In Vitro Tyrosine Kinase Assay-- Primary human breast tissues were dissected and homogenized in cell lysis buffer (same as above). Human cells were starved in serum-free medium, then were unstimulated or stimulated with 10 nM HRG for 10 min, and total protein extracts were prepared as described above (see "Western Blot Analysis"). Next, 1 mg of protein was immunoprecipitated using antibodies against CHK (Lsk, Santa Cruz Biotechnology), Csk (Santa Cruz Biotechnology), Src (clone GD11, Upstate Biotechnology Inc.), and Lyn (Santa Cruz Biotechnology). Washed immunoprecipitates were resuspended in 50 µl of kinase buffer (50 mM Tris-HCl, pH 7.4, 10 mM MnCl2, 0.1% Triton X-100, 1 mM dithiothreitol) containing anti-protease and anti-phosphatase inhibitors (10 µg/ml aprotinin, 10 µg/ml leupeptin, 10 µg/ml pepstatin, 1 mM phenylmethylsulfonyl fluoride, 1 mM sodium orthovanadate), 0.2 mg/ml poly(Glu/Tyr)4:1 (Sigma) as an exogenous substrate, 10 µM unlabeled ATP, and 10 µCi of [gamma -32P]ATP (6000 Ci/mmol, PerkinElmer Life Sciences). After 10 min at 30 °C, the reaction was stopped by adding SDS-sample buffer and resolved on 12% polyacrylamide-SDS gels. The labeled poly(Glu/Tyr) was excised from the gel and radioactivity was counted.

Cell Cycle Analysis-- MCF-7 cells were starved in serum-free medium, then stimulated with 10% fetal bovine serum and harvested by trypsin/EDTA digestion after two washes with phosphate-buffered saline. 1-2 × 106 cells/ml were fixed with 50% ice-cold methanol for 30 min on ice. After centrifugation at 300 × g for 5 min, cell pellets were resuspended in 500 µl of staining solution containing 10 µg/ml propidium iodide (Sigma) and 100 units/ml RNase A (Roche Molecular Biochemicals). Flow cytometric analysis was performed with a FACScan flow cytometer (Becton Dickinson) at the Core Flow Cytometry Facility of the Dana-Farber Cancer Institute (Boston, MA). The percentage of cells in each phase of the cell cycle (G1, S, and G2/M) was calculated with ModFitLT cell cycle analysis software (Verity Software House).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

CHK and Csk Expression and Kinase Activities in Primary Human Breast Tissues and Human Breast Cancer Cell Lines-- To analyze CHK and Csk functions in breast cancer, their expression and kinase activities in normal and malignant breast tissues and cell lines were evaluated. We showed here by RT-PCR and Southern blotting that normal human breast tissues were negative for CHK, whereas breast cancer tissues expressed CHK (Fig. 1A). Csk expression was abundant in both normal breast and tumoral breast tissues (Fig. 1A). In addition, Csk protein was highly expressed while CHK protein was detected at low levels in only some of the tumors by Western blot analyses (Fig. 1B) and immunofluorescence staining (data not shown). We then analyzed the kinase activities of the Csk and CHK proteins extracted from primary human breast carcinomas that were found to express Csk and CHK, as shown by immunohistochemistry (data not shown). We did not detect any CHK activity in the 12 different human breast tumors analyzed, whereas Csk kinase activity was very high (Fig. 1C).


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  		Fig. 1.   CHK and Csk expression and kinase activities in primary human breast tissues. A, poly(A+) RNAs from three human normal breast tissues and three human tumoral breast tissues were analyzed by Southern blotting (SB) using 32P-labeled CHK or Csk cDNA probes (upper and middle panels), followed by hybridization with beta -actin as a control (lower panel). B, expression of Csk and CHK in primary breast tumors and normal breast tissues. Total cell lysates were prepared from normal breast specimens (N) and primary breast cancer tumors (T), and were analyzed for Csk and CHK expression by Western blot analysis using specific antibodies for Csk and CHK, respectively. Actin expression was used as a positive control in these samples. MCF-7/CHK are stably transfected MCF-7 cells overexpressing CHK, and were used as a positive control for CHK expression. C, total protein extracts from three human normal breast tissues and 12 human tumoral breast tissues were immunoprecipitated (IP) with antibodies against CHK or Csk. Normal rabbit serum was used as a control. The tyrosine kinase activity of the immunoprecipitates was determined using poly(Glu/Tyr) as a substrate.

Upon examining human breast cell lines, we found that the human breast cancer cell line MCF-7 expressed CHK as detected by RT-PCR and Southern blotting (Fig. 2A) and Western blotting (Fig. 2B), although the expression level of CHK in MCF-7 cells was about 10-fold less than that found in megakaryocytic cell lines (Dami and MEG-01). Normal epithelial breast cells (MCF-10A and HBL-100) and ZR-75-1 breast cancer cells were negative for CHK expression (Fig. 2, A and B). Analysis of kinase activity showed no activity for CHK, whereas Csk protein was active (Fig. 2C). High Csk expression was observed in all of these cell lines (Fig. 2B).


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  		Fig. 2.   CHK expression and kinase activity in human breast cell lines. A, poly(A+) RNAs from various normal breast cells (MCF-10A, HBL-100) and tumoral breast cells (ZR-75-1, MCF-7) were analyzed by Southern blotting (SB) using a 32P-labeled CHK cDNA probe (upper panel), followed by hybridization with beta -actin as a control (lower panel). Human megakaryocytic cells (Dami, MEG-01) were used as a positive control for CHK expression. B and C, total protein extracts were immunoprecipitated (IP) with antibodies against CHK or Csk. Normal rabbit serum was used as a control. B, immunoprecipitates were analyzed for protein expression by Western blotting (WB) using antibodies against CHK (upper panel) and Csk (lower panel). C, tyrosine kinase activity of the immunoprecipitates (IP) was determined using poly(Glu/Tyr) as a substrate.

To determine whether the lack of CHK kinase activity in breast tumors is due to CHK genetic alterations (point mutations and/or deletions) or due to its low expression levels, we isolated RNA from 10 breast cancer tumors. We then performed CHK sequence analysis and compared the results to that for the wild-type CHK cDNA isolated from megakaryocytes and brain (16). We found no changes/mutations in CHK expressed in breast tumors (data not shown), indicating that lack of CHK kinase activity is due to the low levels of CHK expression in breast tumors.

Our previous study (34) showed that CHK associated with the ErbB-2/neu receptor upon HRG treatment of breast cancer cells and down-regulated ErbB-2/neu-activated Src kinases. To analyze whether Csk also associates with ErbB-2 and down-regulates ErbB-2/neu-activated Src kinases, MCF-7 cells were stimulated with HRG, lysed, and incubated with the purified Csk-SH2 fusion protein. The co-precipitated proteins were analyzed by Western blotting using anti-phosphotyrosine antibody (PY20). No association of ErbB-2 with the Csk-SH2 domain was found (data not shown). Furthermore, no changes were observed in ErbB-2/neu-activated Src kinase activity upon HRG stimulation of MCF-7 cells transfected with the Csk expression vector (data not shown). These data demonstrate that CHK, but not Csk, is involved in HRG-mediated signaling in breast cancer cells.

Generation and Characterization of Stable Transfected Human MCF-7 Breast Cancer Cells Overexpressing CHK-- Because CHK and not Csk is involved in ErbB-2/neu signaling in breast cancer cells, we further characterized the biological effect of CHK overexpression on the proliferation and transformation of transfected MCF-7 cells. To evaluate the effect of overexpression of wild-type kinase-active CHK protein in MCF-7 cells, we generated stable transfected cells overexpressing CHK, either wild-type (wt) or dead-kinase (dk), by point mutation in the lysine of the ATP-binding site of the kinase domain (K262 right-arrow Ala). Positive transfectants were chosen based on their immunoreactivity on Western blots probed with anti-CHK antibody. We selected two clones overexpressing wt CHK (clone #5 and clone #10) and two clones expressing dk CHK (clone #7 and clone #9). Control cells were non-transfected MCF-7 cells (-) and MCF-7 cells transfected with the empty vector (neo).

We first confirmed by Western blot analysis that the level of CHK protein expression was comparable in the different CHK-transfected MCF-7 cells (Fig. 3A). Interestingly, Csk expression was detected in control MCF-7 cells but was not modulated in the CHK-transfected MCF-7 cells. These results show that Csk expression is ubiquitous and not regulated, confirming the results of previous reports in various systems (20, 22, 28).


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  		Fig. 3.   Generation and characterization of stably transfected human MCF-7 breast cancer cells overexpressing CHK. MCF-7 cells were stably transfected with a pcDNA3-neo expression vector containing CHK cDNA either wild-type (wt, clone #5 and clone #10) or dead-kinase (dk, clone #7 and clone #9). Control cells were non-transfected cells (-) and cells transfected with the empty vector (neo). A, total protein extracts were analyzed for protein expression by Western blotting (WB) using antibodies against CHK (upper panel), Csk (middle panel), and actin as an internal control (lower panel). B and C, cells were non-induced (-) or induced with HRG (+). Total protein extracts were immunoprecipitated with antibodies against CHK (B) and Csk (C). The tyrosine kinase activity of the immunoprecipitates was determined using poly(Glu/Tyr) as a substrate. Each experiment is representative of three independent experiments.

CHK and Csk Kinase Activities upon HRG Stimulation of MCF-7 Cells-- Next, we evaluated the kinase activity of the overexpressed CHK (Fig. 3B). Upon stimulation with HRG, CHK kinase activity in MCF-7/CHK(wt) cells was increased (2-fold) compared with values measured in non-induced cells. No significant CHK kinase activity was detected in control MCF-7 cells and MCF-7/CHK(dk) cells. Similar levels of Csk kinase activity were detected in all types of MCF-7 cells, in both the absence as well as the presence of HRG (Fig. 3C), which confirms that Csk is constitutively active and its kinase activity is not affected upon stimulation by HRG and/or by CHK overexpression.

CHK Overexpression Inhibits in Vitro MCF-7 Cell Proliferation, Transformation, and Invasion-- MCF-7/CHK(wt) cell proliferation was not induced upon HRG stimulation, whereas control MCF-7 cells and MCF-7/CHK(dk) cells did proliferate in response to HRG (Fig. 4A). In addition, the anchorage-independent growth of MCF-7/CHK(wt) cells in soft agar was significantly decreased (5- to 6-fold) as compared with control MCF-7 cells and MCF-7/CHK(dk) cells (Fig. 4B).


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  		Fig. 4.   CHK overexpression inhibits in vitro MCF-7 cell proliferation, transformation, and invasion. A, MCF-7 clones were grown in serum-free medium in the absence (dashed line) or presence (solid line) of HRG. The number of viable cells was quantitated by crystal violet staining. The data shown are the mean values of four wells. B, MCF-7 clones were grown in soft agar before counting viable colonies. The data shown are the mean values ± S.D. of four wells. C, MCF-7 clones (as described in Fig. 3) were tested for their ability to invade Matrigel, in the presence (+) or absence (-) of HRG. The data shown are the mean values ± S.D. of three experiments done in triplicate.

It has been reported that ErbB-2/neu is necessary for the induction of carcinoma cell invasion (40) and that MCF-7 cell migration could be induced after HRG stimulation (41). Therefore, we evaluated the biological effect of overexpressed CHK by analyzing the ability of transfected MCF-7 cells to invade Matrigel (Fig. 4C). We confirmed that the invasion of control MCF-7 cells was increased (2-fold) in response to HRG. Upon HRG stimulation, invasion of MCF-7/CHK(wt) cells was significantly inhibited (33% inhibition and p < 0.001) as compared with non-transfected MCF-7 cells. Interestingly, in the presence of HRG, the invasion of MCF-7/CHK(dk) cells was also significantly reduced (24% inhibition and p < 0.001) as compared with non-transfected MCF-7 cells. This suggests that the kinase activity of CHK is required but not sufficient for inhibition of the invasion process.

CHK Overexpression Inhibits MCF-7 Tumor Growth in Nude Mice-- To further demonstrate the anti-tumoral activity of CHK in breast cancer, we evaluated the effect of CHK overexpression on the tumor development of MCF-7 cells grafted in nude mice. CHK-transfected MCF-7 cells were inoculated subcutaneously into mice and tumor size was followed for 60 days (Fig. 5A). The tumor growth of MCF-7/CHK(wt) cells was significantly inhibited as compared with non-transfected MCF-7 cells (97% inhibition and p = 0.047 for clone #5, and 100% inhibition and p = 0.028 for clone #10). No significant tumor reduction was observed for either MCF-7/neo or MCF-7/CHK (dk) cells (p < 0.05). CHK expression was confirmed by Western blot analyses of the tumors obtained at day 60. The level of CHK protein expression was comparable in all tumors obtained from MCF-7/CHK (wt) or (dk) cells (Fig. 5B).


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  		Fig. 5.   CHK overexpression inhibits MCF-7 tumor growth in nude mice. MCF-7 clones were implanted subcutaneously into the mammary fat pad of female athymic nude mice. A, mice were followed for tumor growth for 60 days. Data represent median tumor volumes as a function of time. This experiment is representative of three independent experiments. B, mice were sacrificed at day 60. Total protein extracts from tumors were analyzed for protein expression by Western blotting (WB) using antibodies against CHK (upper panel) and actin as an internal control (lower panel).

CHK Overexpression Down-regulates in Vitro c-Src and Lyn Expression and Kinase Activities-- Next, we investigated the mechanism of breast tumor growth inhibition by CHK. We first evaluated the modulation of CHK substrates, the Src family members. It has been reported that, in MCF-7 cells, two Src family protein-tyrosine kinases could be activated: c-Src kinase (42) and Lyn kinase (43). We observed that, in the absence of HRG stimulation, the level of Src and Lyn protein expression was down-regulated in MCF-7/CHK (wt) cells as compared with control MCF-7 cells and MCF-7/CHK(dk) cells (Fig. 6A). To induce CHK kinase activity, transfected MCF-7 cells were stimulated with HRG. Src kinase activity was significantly inhibited (62% inhibition) in MCF-7/CHK(wt) cells (Fig. 6B), as well as Lyn kinase activity (78% inhibition) (Fig. 6C). In the absence of HRG stimulation, no Src and Lyn kinase activities were observed (data not shown).


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  		Fig. 6.   CHK overexpression down-regulates in vitro c-Src and Lyn expression and kinase activities. A, immunoprecipitates of MCF-7 clones, in the absence of HRG stimulation, were analyzed for protein expression by Western blotting (WB) using antibodies against Src (upper panel) and Lyn (middle panel). Total protein extracts were also analyzed by Western blotting using antibodies against actin as an internal control (lower panel). B and C, MCF-7 clones were induced with HRG, then total protein extracts were immunoprecipitated (IP) with antibodies against Src and Lyn. Tyrosine kinase activity of Src (B) and Lyn (C) immunoprecipitates was determined using poly(Glu/Tyr) as a substrate. The data shown are the mean values ± S.D. of duplicate samples. Each experiment is representative of three independent experiments.

CHK Overexpression Delays in Vitro MCF-7 Cell Entry into Mitosis-- We further investigated the mechanism of CHK action occurring after the down-regulation of Lyn kinase. Because it has been previously demonstrated that Src family kinases are required for cell division to occur (44) and are specifically required at the transition from the G2 phase to mitosis in the cell cycle (45), we therefore investigated whether the overexpression of CHK might modulate cell cycle kinetics. A significant delay in the entry to S-phase (12 h) and an increase in G2/M phase (2-fold) were observed with MCF-7/CHK(wt) cells as compared with the non-transfected MCF-7 cells (Table I).

                              
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Table I
CHK overexpression delays in vitro MCF-7 cell entry into mitosis
MCF-7/CHK (wt) clone #10 and control MCF-7 cells were stimulated with FBS and then analyzed by flow cytometry at the indicated time points. The percentage of cells in each phase of the cell cycle (G1, S, and G2/M) was calculated. Similar results were obtained with MCF-7/CHK (wt) clone #5 cells (data not shown).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Little is known about the role(s) of CHK in cellular physiology. Unlike the ubiquitous expression of Csk in both normal and tumoral breast tissues, CHK expression was low in primary human breast cancer tissues and was not detected in normal breast tissues. In this study, we observed that the CHK protein expressed in the MCF-7 human breast carcinoma cell line and in primary human breast cancer tissues was kinase-inactive due to the low level of CHK protein expression, whereas Csk was kinase-active. Sequence analysis of CHK expressed in breast tumors demonstrated no genetic alterations (mutations and/or deletions) in the CHK gene. However, unlike Csk, CHK kinase activity was significantly induced upon HRG stimulation, and CHK was found to associate through its SH2 domain with ErbB-2/neu and to down-regulate ErbB-2/neu-activated Src kinases in breast cancer cells upon HRG stimulation (33-34). Our results indicate that CHK, and not Csk, is involved in ErbB-2- and HRG-mediated signaling in breast cancer cells. Recently, substantial elevation of Csk protein levels in human carcinomas was reported (46). Furthermore, up to 20% of patients with carcinomas had high affinity auto-antibodies against Csk, demonstrating that Csk acts as an auto-antigen (46). This overexpression of Csk was correlated with a strong increase in Src activity, which suggests that Csk cannot regulate Src activity in these carcinomas. These results are in agreement with our results demonstrating that CHK expression is up-regulated in breast cancer cells and that CHK, but not Csk, is involved in the inhibition of ErbB-2/neu-activated Src kinases upon HRG treatment.

To further analyze CHK function in breast cancer cells upon HRG stimulation, we evaluated the biological effect of overexpressing a wild-type CHK protein in MCF-7 cells. We generated stable transfected human MCF-7 breast carcinoma clones that overexpressed CHK. We showed that overexpression of wild-type (wt) CHK inhibited MCF-7 cell proliferation and transformation (Fig. 4). These effects were dependent on CHK kinase activity, because they were detected only after stimulation with HRG and were not observed with the dead-kinase (dk) CHK. Interestingly, overexpression of both wt and dk CHK forms inhibited MCF-7 cell invasion upon HRG stimulation (Fig. 4), suggesting that the kinase activity of CHK is required but not sufficient for inhibition of the invasion process. It has been reported that overexpression of CHK suppressed VLA5 integrin-mediated cell spreading and that this suppression was dependent upon both the CHK SH3 domain, which is responsible for membrane anchoring, and CHK kinase activity, which is responsible for Lyn kinase inactivation (26). This suggests that CHK can inhibit cell invasion via both its SH3 domain and its kinase activity. Moreover, overexpression of wt CHK dramatically inhibited MCF-7 tumor growth in nude mice (Fig. 5). This inhibition was not observed with the dead-kinase CHK. Altogether, our results demonstrate that CHK inhibits both invasion and growth of human breast carcinoma cells.

We then investigated the mechanism of breast tumor growth inhibition by CHK. We showed that, unlike the constitutive activity of Csk, CHK kinase activity was induced upon HRG stimulation (Fig. 3). This increased activity correlated with a decrease in Src and Lyn protein expression and activity (Fig. 6). In addition, overexpression of CHK led to G2/M cell cycle arrest, which delayed cell entry into mitosis (Table I). Src family kinases are activated in ErbB-2/neu-induced mammary tumors (4) through direct binding to the ErbB-2/neu receptor (11), and we have previously shown that, upon HRG stimulation, CHK binds to the ErbB-2/neu receptor and down-regulates ErbB-2/neu-activated c-Src kinases (33, 34). In addition, it has been reported that (i) Src family kinases are involved in cell proliferation and transformation induced in response to growth factor stimulation (12), (ii) Src family kinase activation by ErbB-2/neu leads to attachment-independent growth (47) and invasion (48) of human breast epithelial cells, and (iii) Src family kinases are specifically required at the transition from the G2 phase to mitosis in the cell cycle (45). Previous reports as well as our results demonstrate that the mechanism of inhibition of breast tumor growth by CHK is through the inhibition of ErbB-2/neu-mediated Src family kinase activation, leading to delayed cell entry into mitosis, and inhibition of attachment-independent growth and cell invasion.

New therapeutic interventions for breast cancer are under intensive investigation. Protein-tyrosine kinases play a fundamental role in signal transduction pathways, and dysregulated tyrosine kinase activity has been observed in many proliferative diseases (49). Therefore, there is an increasing interest in targeting cell surface receptor tyrosine kinases as well as non-receptor tyrosine kinases (50, 51). Substantial evidence indicates that the ErbB-2/neu receptor plays an important role in breast cancer (3). Therefore, the effort to inhibit ErbB-2/neu activity is an attractive approach in breast cancer therapy. A recombinant humanized monoclonal antibody trastuzumab (Herceptin, Genentech Inc., San Francisco, CA), directed against the extracellular domain of ErbB-2, was shown to inhibit the proliferation of breast cancer cells (52, 53) and is under evaluation in clinical trials (54). Another recent approach is to suppress ErbB-2/neu overexpression, leading to inhibition of tumorigenesis (55). In addition, Src family kinases have also been shown to play a fundamental role in breast cancer and to be activated in ErbB-2/neu-induced mammary tumors (4). Therefore, inhibition of Src kinases is another promising approach in breast cancer therapy. Tyrphostins are synthetic compounds that have been described as tyrosine kinase inhibitors (56). It has been reported that treatment with tyrphostins inhibited c-Src kinase activity in vitro (57). Thus, protein-tyrosine kinases are potential targets for the design of new therapeutic agents against cancer. In this study, we have shown that CHK down-regulates HRG-mediated ErbB-2/neu and Src family kinases and that overexpression of wild-type CHK protein down-regulates breast tumor growth. Further studies will investigate whether overexpression of CHK might be an alternative strategy for the inhibition of ErbB-2/neu and/or Src family kinase pathways and will ultimately elucidate the potential role for CHK in breast cancer gene therapy.

    ACKNOWLEDGEMENTS

We thank Yigong Fu for construction of the mutated CHK (K262A)-pcDNA3 vector; Bijia Deng and Yiming Ding for technical assistance with in vivo studies; Jakub Golab for help with statistical analyses; Sheila Zrihan-Licht for help with cell cycle analysis; Mark X. Sliwkowski (Department of Protein Chemistry, Genentech Inc., San Francisco, California) for providing heregulin; Dan Kelley for help with preparation of the figures; and Janet Delahanty for editing of the manuscript.

    FOOTNOTES

* This work was supported in part by National Institutes of Health Grants CA 76226 and CA 87290 (to H. A.), by U.S. Army Medical Research and Material Command Grants DAMD 17-98-1-8032 and DAMD 17-99-1-9078 (to H. A.), by Experienced Breast Cancer Research Grant 34080057089 (to H. A.), by the Milheim Foundation (to H. A.), and by the Massachusetts Department of Public Health (to H. A.) and (to C. B.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

This paper is dedicated to Charlene Engelhard for her continuing friendship and support for our research program.

This work was done during the term of an established investigatorship from the American Heart Association (to H. A.). To whom correspondence should be addressed: Division of Experimental Medicine, Beth Israel Deaconess Medical Center, Harvard Institutes of Medicine, 4 Blackfan Circle, Boston, MA 02115. Tel.: 617-667-0073; Fax: 617-975-6373; E-mail: havraham@caregroup.harvard.edu.

Published, JBC Papers in Press, July 9, 2001, DOI 10.1074/jbc.M104209200

    ABBREVIATIONS

The abbreviations used are: EGF, epidermal growth factor; Csk, carboxyl-terminal src kinase; CHK, Csk homologous kinase; HRG, heregulin; SH, Src homology; IL-1, interleukin-1; wt, wild-type; dk, dead-kinase; RT-PCR, reverse transcription-polymerase chain reaction.

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EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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