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Cell and Tumor Biology |
Departments of 1 Surgical Oncology and 2 Molecular and Cellular Oncology, University of Texas M.D. Anderson Cancer Center, Houston, Texas
Requests for reprints: Dihua Yu, Department of Surgical Oncology, University of Texas M.D. Anderson Cancer Center, Unit 107, Houston, TX 77030. Phone: 713-792-3636; Fax: 713-794-4830; E-mail: dyu{at}mdanderson.org.
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Abstract |
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Key Words: breast cancer • metastasis • Src • ErbB2 • mTOR • calpain
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Introduction |
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30% of human breast cancers (3) and in many other cancer types (4). Patients with ErbB2-overexpressing breast cancer have substantially lower survival rates and shorter times to relapse than patients without the overexpression. Moreover, many clinical and laboratory investigations have shown that overexpression of ErbB2 leads to increased breast cancer metastasis (5, 6). Results from our laboratory also showed that ErbB2 overexpression enhances the intrinsic metastatic potential of human breast cancer cells (7).
The ErbB2 RTK has intrinsic tyrosine kinase activity (1, 6). Activation of the ErbB2 tyrosine kinase leads to autophosphorylation of tyrosine residues in its carboxyl-terminal domain. The phosphorylated tyrosine residues then function as docking sites for ErbB2 downstream signaling molecules (6, 8). Previous studies showed that kinase-activated ErbB2 physically associates with Shc (9), Grb-7 (10), Ras-GTPase-activating protein (11), phospholipase C
(11, 12), the 85-kDa regulatory subunit (p85) of phosphatidylinositol 3-kinase (12, 13), Src (14, 15), ß-catenin (16), Csk-homologous kinase (17), Erbin (18), Ralt (19), c-Cbl (20), and p34Cdc2 (21). However, the overall picture of how ErbB2 exerts its function through these signaling molecules is unclear, and the signaling events responsible for ErbB2-mediated cancer metastasis remain elusive. Because most patients with ErbB2-overexpressing breast cancer die from metastasis, unraveling the molecular mechanisms and signaling events that underlie ErbB2-mediated metastasis is extremely important and may facilitate the development of therapies or prevention strategies for patients with ErbB2-overexpressing tumors.
The human c-Src proto-oncogene encodes an intracellular tyrosine kinase, pp60c-Src (22, 23). c-Src was originally identified as a homologue of the transforming gene of the Rous sarcoma virus (24) and is the prototype of a family of highly conserved genes that includes yes, fyn, fgr, lyn, lck, hck, blk, and yrk. Src family proteins have been implicated in many signal transduction pathways and a wide variety of cellular functions (25). Activation of Src kinase has been linked to the development of many human neoplasias, especially those of the colon, breast, lung, and pancreas (26). Most of the previous Src studies focused on the deregulation of Src kinase activity but not the altered expression levels of Src protein. The deregulated Src protein synthesis and stability in mediating malignant phenotypes of cancer cells have been largely neglected.
Tumors and cell lines that overexpress ErbB2 have been reported to contain activated Src family kinases, especially pp60c-Src (14, 15, 27). Although these previous studies suggest that Src activation might play a role in the malignant phenotypes induced by ErbB2, whether Src is important in ErbB2-mediated breast cancer metastasis has not been investigated, and the mechanism by which ErbB2 activates Src has not been well established.
To elucidate the signaling pathways downstream of ErbB2 that contribute to ErbB2-mediated breast cancer metastasis, we expressed wild-type (wt) or mutant ErbB2 proteins with altered signaling capacity in the MDA-MB-435 human breast cancer cell line that expresses low levels of ErbB2. We then examined the metastatic potentials of these cells and their relationship with the activation of ErbB2 downstream signals that might be involved in metastasis. We found that ErbB2 activated Src kinase through the up-regulation of the Src protein level via increasing Src protein synthesis and stability. Moreover, we showed that increased Src activities were required for ErbB2-mediated breast cancer metastasis.
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Materials and Methods |
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Design and Construction of Small Interfering RNA. Small interfering RNAs (siRNA) were designed to target ErbB2-specific sequences. We synthesized two oligonucleotides: 5'-AAGTACACGATGCGGAGACTGCCTGTCTC-3' and 5'-AACAGTCTCCGCATCGTGTACCCTGTCTC-3'. The Silencer siRNA Construction kit (Ambion, Austin, TX) was used to obtain the double-stranded siRNA according to the manufacturer's instruction.
Cell Lines and Cell Cultures. The MDA-MB-435 human breast cancer cell line, 435.eB cells (which are MDA-MB-435 cells stably expressing wt ErbB2), and 435.neo cells were obtained, established, and cultured as reported previously (28). The ErbB2 mutants were subcloned from the pSV2 vectors into pcDNA3 expression vectors (Invitrogen, Carlsbad, CA) in which transcription is driven by a cytomegalovirus promoter. The resulting plasmids were transfected into MDA-MB-435 cells as described (29), and the stable transfectants were selected by G418 (geneticin, Life Technologies, Rockville, MD). Western blot was done to identify transfectants that expressed the ErbB2 protein. Stable wt ErbB2 transfectants of the human breast cancer cell line MDA-MB-231 were established using the same method. The dominant-negative mutant sequence of Src (DNSrc) was subcloned from the expression vector pcR295 into the pIRESpuro2 vector (Clontech, Palo Alto, CA). The resulting plasmid (DNSrc) was transfected into 435.neo or 435.eB cells, and the transfectants were selected using puromycin (Clontech). HA-tagged ubiquitin expression vector (pCGN-Ub) was constructed and expressed as described previously (30). All breast cancer cells used in this study were grown in DMEM and Ham's F-12 (DMEM/F-12, Life Technologies). Osteosacoma cell line Saos-2 was cultured in RPMI 1640 (Life Technologies).
Western Blot and Immunoprecipitation Analyses. Western blot and immunoprecipitation analyses were done as described previously (31). The protein inputs in the Western blot and immunoprecipitation analyses were normalized by loading the equal amount of total protein lysates onto the SDS-PAGE gels. The nonradioactive signals were quantified by Alpha Imager 2000 (Alpha Innotech, San Leandro, CA). The radioactive signals were quantified by PhosphoImager (Molecular Dynamics, Sunnyvale, CA).
In Vitro Cell Invasion Assay. Cell invasion was assayed as described previously (7).
In Vivo Metastatic Survival Rates. Single-cell suspensions of each cell line were injected into the lateral tail veins of 8-week-old female severe combined immunodeficient (ICR-SCID) mice (1 x 106 cells per mouse, Harlan, Madison, WI). The injected mice were maintained under identical conditions and were monitored regularly. Once a mouse became moribund, euthanasia and necropsy were done, and a veterinary pathologist was consulted to determine the cause of the animal's death. The times of death for the mice that died of metastasis were plotted. Survival curves were drawn and analyzed using Prism software (GraphPad Software, San Diego, CA). The survival curves were compared by the Mantel-Haenszel log-rank test. All animal experiments in this study were done under the animal use guidelines of the NIH (Bethesda, MD) and the Animal Care and Use Committee of the University of Texas M.D. Anderson Cancer Center (Houston, TX).
Src Kinase Assay. Cells were lysed and immunoprecipitated with anti-Src antibodies. The immunoprecipitates were washed thrice with immunoprecipitation buffer and twice with a kinase buffer containing 10 mmol/L Tris-HCl (pH 7.4), 150 mmol/L NaCl, 10 mmol/L MgCl2, 1 mmol/L DTT, and 0.5 mmol/L orthovanadate. Kinase activity was assayed by incubating the immunoprecipitates with 40 µL kinase buffer containing 250 µg/mL enolase, 2 µCi [-32P]ATP, and 3 µL of 100 mmol/L ATP. After 30 minutes at 37°C, the reaction was terminated by adding 15 µL of 6x SDS sample buffer and boiling for 5 minutes. The samples were subjected to SDS-PAGE. The [-32P]ATP-labeled enolase was visualized by autoradiography.
RNA Interference. BT474 breast cancer cells were cultured to 30% confluence and then transfected with various concentrations of ErbB2 siRNA. After 3 hours of transfection, cells were incubated with serum-containing medium and cell lysates were collected for Western blot analysis 48 hours after transfection. A siRNA concentration of 66.7 nmol/L was able to significantly decrease ErbB2 protein levels compared with that in the control GFP siRNA- or mock-transfected cells.
Assay of Protein Half-life by Pulse-Chase Experiment. The cells were washed thrice with [35S]methionine-free DMEM (Life Technologies) supplemented with 3% fetal bovine serum. The cells were incubated for 1 hour with the wash medium containing 500 µCi/mL [35S]methionine (Amersham Pharmacia, Piscataway, NJ). The cells were washed thrice and incubated in normal growth medium. At the different chase time points, the cells were lysed in immunoprecipitation buffer, and Src protein was immunoprecipitated by the Src antibody. The immunoprecipitates were subjected to SDS-PAGE, and radiolabeled Src was detected by autoradiography.
Protein Synthesis Assay. The cells were washed thrice with [35S]methionine-free DMEM (Life Technologies) supplemented with 3% fetal bovine serum and then incubated for 1 or 4 hours with the wash medium containing 500 µCi/mL [35S]methionine. Cell lysates were extracted and equal amounts of protein lysates were subjected to immunoprecipitation with the antibody against Src or ß-actin, and the immunoprecipitates were analyzed by SDS-PAGE and radiolabeled proteins were detected by autoradiography.
Experimental Metastasis Assays. We injected 1 x 106 cancer cells into the tail veins of female ICR-SCID mice. On the same day of the injections, the mice were injected i.p. with 1 mg/kg body weight of Src kinase inhibitor PP2 or its solvent, 10% DMSO-PBS. Three days later, the mice were injected with a second dose of PP2 or 10% DMSO-PBS. The injection schedules were then changed to once a week, and the injections continued for 12 weeks. The mice were euthanized 90 days later, and their lungs were instilled with India ink. Metastatic lung nodules >0.5 mm in diameter were counted. Statistical analyses were done by Student's t test.
The experimental metastasis assay by i.v. injection of the 435.eB or eB.DNSrc was done and analyzed similar to the above described, except that 2 x 106 cancer cells were injected, mice were not treated with PP2, and mice were euthanized 60 days after injection.
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To determine the impact of ErbB2 activation on breast cancer metastasis, we examined MDA-MB-435 cells, 435.neo cells, and wt or mutant ErbB2 transfectants for their metastatic potential in vitro and in vivo. An invasion assay showed that 435.eB and V659E cells were highly invasive compared with MDA-MB-435, 435.neo, and K753M cells (Fig. 1B), which correlated with increased ErbB2 kinase activities (Fig. 1A). To test the metastatic potential of these cells in vivo, we injected the cells into the lateral tail veins of immunodeficient ICR-SCID mice. The mice later died of metastases, and the metastatic survival rates of the mice were recorded. The mice injected with 435.eB or V659E cells had significantly lower metastatic survival rates than those injected with MDA-MB-435 and 435.neo cells (435.eB versus 435.neo, P = 0.0420; V659E versus 435.neo, P = 0.0252; Fig. 1C). The metastatic survival rates of mice injected with K753M cells were similar to those of mice injected with MDA-MB-435 and 435.neo cells (Fig. 1C). In summary, 435.eB and V659E cells with high ErbB2 kinase activities were highly metastatic, whereas MDA-MB-435, 435.neo, and K753M cells with low kinase activities were not. These results show that ErbB2 kinase activity plays a critical role in ErbB2-mediated metastasis in these breast cancer cells.
Up-Regulation of Src Protein by ErbB2. We next investigated multiple ErbB2 downstream signaling pathways that may contribute to ErbB2-mediated metastasis, and we focused on Src because it was tightly regulated by altered ErbB2 RTK activities and closely correlated with the metastatic potential of our panel of breast cancer cells (see below). When we compared Src activity in MDA-MB-435 cells, 435.neo cells, and wt and mutant ErbB2 transfectants by immunocomplex kinase assay (Fig. 2A), we found that the Src kinase was activated at dramatically higher levels in 435.eB and V659E cells than in the control MDA-MB-435, 435.neo, and K753M cells. Thus, activation of ErbB2 RTK by overexpression or activating mutation can lead to Src activation in these breast cancer cells.
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To further confirm that ErbB2 regulates Src protein expression levels, the ErbB2 high-expressing breast cancer cell line BT474 was transfected with siRNA to ErbB2 (Fig. 2D, left). Immunoblotting results showed that ErbB2 siRNA down-regulated ErbB2 expression in BT474 cells and subsequently reduced Src protein levels. In addition, compared with 435.eB, the Src protein level was reduced in the 435eB.R spontaneous revertant, a 435.eB derivative that partially lost the transfected erbB2 gene and expresses lower levels of ErbB2 (Fig. 2D, middle). Moreover, elevated Src protein expression could also be observed in the wt ErbB2-transfected breast cancer cell line MDA-MB-231 (231.eB) compared with their parental cells (MDA-MB-231; Fig. 2D, right). Furthermore, by immunohistochemistry analysis, we studied Src protein expression levels in breast cancer patient samples, which were strongly correlated with ErbB2 expression levels (data not shown) as reported previously (36). These data show that Src protein expression is regulated by ErbB2 in breast cancer cells. Taken together, we conclude that ErbB2 activation can up-regulate Src at the post-transcriptional level and thereby increase Src protein and kinase activity in ErbB2-activated human breast cancer cells.
ErbB2 Increases Src Protein Synthesis by Activating the Akt/Mammalian Target of Rapamycin/4E-BP1 Protein Translation Pathway. To investigate the mechanism of Src protein up-regulation by ErbB2, we first tested whether the rate of Src protein synthesis was greater in ErbB2-overexpressing cells than in ErbB2 low-expressing cells. MDA-MB-435 and 435.eB cells were cultured with medium containing [35S]methionine to metabolically label the newly synthesized proteins for 1 or 4 hours. Src proteins from the cell lysates were immunoprecipitated with a Src-specific antibody, analyzed by SDS-PAGE, and visualized by autoradiography. The results showed that ErbB2-overexpressing 435.eB cells had higher levels of [35S]methionine-labeled Src protein than MDA-MB-435 cells at both 1- and 4-hour 35S labeling (Fig. 3A, left and middle), although the overall rate of incorporation of [35S]methionine was similar between the two cell lines (data not shown). Meanwhile, immunoprecipitation on the same [35S]methionine-labeled cell extracts with the ß-actin antibody and subsequent analysis showed that the ß-actin synthesis rates in MDA-MB-435 and 435.eB cells were similar (Fig. 3A, right). These results, in conjunction with the observation that the Src protein degradation during the 4-hour [35S]methionine labeling was minimal in these cells (see Fig. 4A), suggest that ErbB2 preferentially increased the Src protein synthesis rate in ErbB2-overexpressing 435.eB cells.
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To test whether activation of the mTOR pathway was responsible for increasing 4E-BP1-Ser65 phosphorylation and ErbB2-mediated Src protein synthesis, we treated MDA-MB-435 and 435.eB cells with rapamycin, which is a mTOR-specific inhibitor (39). Inhibition of mTOR by rapamycin effectively reduced 4E-BP1-Ser65 phosphorylation (Fig. 3C). Notably, MDA-MB-435 cells had a minimal reduction in Src protein levels after rapamycin treatment, whereas ErbB2-overexpressing 435.eB cells had a evident reduction in Src protein levels (Fig. 3D), indicating that activation of the Akt/mTOR/4E-BP1 pathway by ErbB2 in 435.eB cells is involved in the enhanced Src protein synthesis. To further test this hypothesis, we treated the cells with or without rapamycin and labeled the cells with [35S]methionine for 4 hours. Immunoprecipitation of Src protein was done, and the immunoprecipitates were analyzed by SDS-PAGE. The results showed that without rapamycin more [35S]methionine-labeled Src protein was detected in 435.eB cells than that of in MDA-MB-435 cells (Fig. 3A, left and middle). However, in rapamycin-treated cells, the [35S]methionine-labeled Src protein in 435.eB cells was reduced to a level similar to that in MDA-MB-435 cells (Fig. 3A, left), indicating that ErbB2-dependent mTOR activation is required for ErbB2-promoted Src protein translation.
ErbB2 Stabilizes Src by Inhibiting Calpain-Mediated Src Degradation. To test whether the elevated Src protein level detected in ErbB2-activated cells may also result from reduced Src protein degradation, pulse-chase analyses were done to test whether ErbB2 increases the stability of Src protein. MDA-MB-435 and 435.eB cells were incubated with [35S]methionine for 1 hour. Free [35S]methionine was washed out, and Src protein was chased at different time points by immunoprecipitation with the Src-specific antibody and visualized by autoradiography. The results showed that the Src degradation was slower in 435.eB cells than in MDA-MB-435 cells and that the Src protein half-life was longer in 435.eB cells (52 hours) than in MDA-MB-435 cells (20 hours; Fig. 4A). Thus, activation of ErbB2 leads to Src protein stabilization, which can also contribute to the increased Src protein levels in the ErbB2-activated cells.
It has been shown that Src could be degraded by the proteasome-ubiquitin-dependent pathway (40). To investigate the mechanism of Src stabilization by ErbB2, we investigated whether ErbB2 activation inhibits ubiquitination-mediated Src protein degradation. A HA-tagged ubiquitin-expressing construct was transfected into MDA-MB-435 and V659E cells. The expression of the HA-tagged ubiquitin was confirmed by Western blot with antibody against HA (Fig. 4B). To detect the ubiquitination of Src, cells were treated or untreated with proteasome inhibitor MG-132, and immunoprecipitation with the Src-specific antibody and subsequent Western blot with the anti-HA antibody were done (Fig. 4B, left). Src ubiquitination could not be detected under these experimental conditions. Contrarily, control experiment showed that ubiquitinated Met, a control protein, was readily detected in the HA-ubiquitin-expressing osteosarcoma cell line Saos-2 with MG-132 treatment (Fig. 4B, right), indicating that the assay is sufficiently sensitive for detecting the ubiquitination of proteins. Next, we examined whether blocking the proteasome pathway with MG-132 will stabilize Src in MDA-MB-435 and V659E cells. Cell lysates were collected from MG-132-treated or untreated cells and immunoblotted with Src and cyclin B1 antibodies. The results showed that MG-132 treatment led to an obvious accumulation of the control protein cyclin B1 that was well known to be degraded through proteasome pathway (41). In contrast, MG-132 treatment did not lead to Src protein accumulation in either of the cell lines (Fig. 4C). This, in conjunction with the failure of detecting Src protein ubiquitination, suggests that the ubiquitin-proteasome pathway may not be a significant pathway for Src degradation in these cells.
Calpains belong to a family of Ca2+-dependent proteases and have been implicated in a wide range of cellular functions, including apoptosis, proliferation, and migration (42). Previous reports suggested that calpain could degrade Src and lead to decreased Src kinase activity in human platelets (43, 44). Therefore, we hypothesized that in ErbB2-overexpressing breast cancer cells ErbB2 may inhibit calpain-mediated Src protein degradation, which could be involved in stabilizing Src. We first examined whether Src was a substrate of calpain in our cell system. Protein lysates from 435.eB cells were incubated with or without 5 or 50 µU purified calpain under various conditions, and the Src protein level was determined by Western blot analysis (Fig. 4D, top). In the presence of 5 mmol/L EGTA, a chelator of Ca2+, there was no obvious calpain activity toward Src. In the absence of added Ca2+, calpain activity toward Src was minimal. However, in the presence of 5 mmol/L CaCl2, calpain caused a dramatic Src protein cleavage into smaller fragments in a concentration-dependent manner. In contrast, immunoblotting with antibody against 14-3-3, a control protein, did not detect 14-3-3 degradation by calpain (Fig. 4D, top), indicating that the proteolytic cleavage is Src specific. To examine the time course of calpain-mediated Src degradation, protein lysates from 435.eB cells were treated with 50 µU calpain in the presence of 5 mmol/L CaCl2 for different times (Fig. 4D, bottom). Immunoblotting with Src antibody showed that the total Src protein detected by the antibody was reduced with prolonged incubation, suggesting that calpain can trigger complete degradation of Src. These data indicate that Src could be a substrate of calpain in these cells.
To test whether an inhibition of calpain-mediated Src degradation was involved in ErbB2-mediated Src stabilization, we incubated MDA-MB-435 and 435.eB cells in the presence or absence of cycloheximide to block protein synthesis or cycloheximide plus calpain-specific inhibitor N-acetyl-leucyl-leucyl-norleucinal. At different time points, protein lysates were collected and the Src protein level was determined by Western blot analysis (Fig. 4E). In the presence of the calpain inhibitor N-acetyl-leucyl-leucyl-norleucinal, degradation of Src protein was delayed in both MDA-MB-435 and 435.eB cells compared with cells treated with cycloheximide alone (Fig. 4E, left). Notably, the delay of Src protein degradation was more evident in MDA-MB-435 cells than in 435.eB cells, suggesting that Src degradation by the calpain pathway was less active in 435.eB cells than in MDA-MB-435 cells most likely as a result of ErbB2 activation. These findings were supported by using another calpain-specific inhibitor N-acetyl-leucyl-leucyl-methional, which has been shown to have little inhibition of proteasome-mediated protein degradation (ref. 41; Fig. 4E, right). Thus, in addition to up-regulating the Src protein level by increasing Src protein synthesis via activation of the Akt/mTOR/4E-BP1 pathway, ErbB2 may also stabilize Src protein via inhibition of calpain-mediated Src degradation.
Src Plays a Crucial Role in ErbB2-Mediated Breast Cancer Metastasis. To assess the role of Src activation in ErbB2-induced breast cancer metastasis, we tested whether PP2, a Src-specific inhibitor (45), can inhibit the invasiveness of the ErbB2 transfectants that contain activated Src. The cells were treated with PP2 or its control chemical PP3 and transferred into transwell units for chemoinvasion assays. PP2 dramatically inhibited the basement membrane invasion abilities of ErbB2-overexpressing, Src-activated 435.eB and V659E cells and to a much lesser extent the invasion abilities of 435.neo control cells (Fig. 5A). Moreover, expression of a DNSrc in 435.neo and 435.eB cells reduced their invasive abilities in the chemoinvasion assay (Fig. 5B). Similar to the treatment with PP2, the inhibition of invasiveness by the DNSrc was more dramatic in 435.eB cells compared with 435.neo cells. These results show that Src kinase plays a critical role in ErbB2-induced cancer cell invasion. We then did experimental metastasis assays to determine the role of Src in ErbB2-mediated metastasis in vivo. The 435.neo cells, which had lower ErbB2 expression and Src kinase activity, and the V659E cells, which overexpressed activated ErbB2 and had a higher Src kinase activity, were injected into the tail veins of female ICR-SCID mice. The mice were sacrificed 90 days after the injection. Metastatic lung nodules were counted and statistically analyzed by Student's t test (Table 1). Consistent with the results from the in vitro invasion assay (Fig. 5A and B), PP2 dramatically inhibited the metastatic potential of ErbB2-activated V659E cells (P < 0.05) but did not significantly inhibit metastasis of 435.neo control cells (P > 0.05). To further investigate the role of Src in ErbB2-mediated cancer cell metastasis, another experimental metastasis assay was done to compare the metastatic potentials of 435.eB cells and 435.eB cells expressing a DNSrc (eB.DNSrc). The result showed that compared with the mice injected with 435.eB cells mice injected with eB.DNSrc cells formed fewer pulmonary nodules (P < 0.05; Fig. 5C), and their lungs weighed less (P < 0.01; Fig. 5D). These in vitro and in vivo results firmly show that Src plays a critical role in ErbB2-mediated human breast cancer cell invasion and metastasis.
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Understanding the molecular mechanisms of ErbB2-mediated metastasis is important for the development of new therapies to treat metastases derived from ErbB2-overexpressing breast cancer. ErbB2 overexpression can be detected in 50% to 60% of high-grade ductal carcinoma in situ breast cancers (46). Interestingly, activation of ErbB2 receptor in preformed mammary acini grown in three-dimensional cell cultures has led to altered mammary acini structures that share properties with ductal carcinoma in situ. No invasive phenotypes were found in these altered acini structures (47). The findings suggest that activation of ErbB2 can induce early stages of mammary carcinogenesis but is not sufficient to induce invasive phenotypes (47). It has been suggested that additional genetic or epigenetic events are needed for mammary epithelial cells to acquire invasive properties (47). The MDA-MB-435 is a highly aggressive metastatic breast cancer cell line and it harbors multiple genetic or epigenetic alterations that may facilitate the development of metastasis (48).
Little was known on the role of Src in ErbB2-induced breast cancer metastasis and on the mechanisms of Src regulation by ErbB2. In this study, we showed that Src plays a critical role in ErbB2-mediated breast cancer invasion and metastasis. To our knowledge, this study reports the first experimental evidence that the higher metastatic potential of ErbB2-overexpressing cancer cells is mediated at least in part through the activation of Src. Therefore, this study provides a rationale for targeting Src as a therapeutic strategy for the treatment or prevention of ErbB2-mediated breast cancer metastasis. It may also have important implications in treating other RTK-mediated malignant cancer behaviors.
Tumor samples from a substantial portion of colon cancers (49, 50) and other cancer types (50), including 48% of breast cancers (51), were shown to have elevated Src protein levels in cancer cells compared with normal cells. However, few studies were devoted to investigating the role and mechanisms of deregulated Src protein synthesis and stability in mediating malignant phenotypes. Our study revealed that the up-regulation of Src protein is an important mechanism for Src activation in ErbB2-overexpressing breast cancer cells. This mechanism may also be exploited by other growth factor receptors or other types of cancer cells in addition to ErbB2 and breast cancer cells. Based on our findings, the overexpressed Src protein in the cancer patient samples may conceivably be due at least in part to growth factor receptor–induced protein up-regulation. The new mechanisms revealed in this study may also have important clinical implications for designing Src-targeting therapies. For example, drug designers may enhance Src blocking efficiency by inhibiting Src protein synthesis and/or inducing Src protein degradation in addition to inhibiting Src kinase activity.
In cancer research, studies on how signaling pathways regulate the transcriptional control of gene expression have been pursued extensively. In contrast, fewer studies have focused on how these pathways control other fundamental processes (52, 53). Recently, modulating protein translational control by signaling pathway activation has emerged as another important regulation mechanism (38, 52–55). Our study provides an excellent example of how signaling molecules can be regulated at the protein level by modulating their protein translational mechanism. To our knowledge, this is the first report that ErbB2 activates the mTOR/4E-BP1 translation pathway and thereby increases Src protein synthesis. Notably, ErbB2 preferentially promotes the translation of Src, which is a typical growth-regulated protein, but not the translation of ß-actin, which is a typical constitutive protein in most cells (Fig. 3A). This is consistent with previous reports that the activation of mTOR preferentially up-regulates the translation of mRNAs of growth-regulated proteins (56).
Previous reports suggested that Src might be a substrate of the Ca2+-dependent protease calpain in human platelets (43, 44) . However, calpain inhibitors or activators were used to modulate Src protein in these previous studies, and it was not clear whether Src was a direct substrate of the calpain protease. By using purified calpain, we provided another evidence to support that Src might be a substrate of calpain (Fig. 4D). Furthermore, in our study, we discovered a biological function for calpain-mediated Src degradation as a way of regulating Src signaling by RTK, which might play an important role in ErbB2 (or other RTKs)–enhanced cancer metastasis. These were not addressed by previous studies. However, the following questions remain to be answered: whether ErbB2 directly or indirectly modulates calpain protease activity, what the mechanism of this modulation is, and what other biological functions this modulation may exert. Given the fact that both ErbB2 and Src play important roles in cancer development and progression and the ubiquitous expression and important functions of calpain protease in the human body, future investigations of these questions will lead to a better understanding of how cancer metastasis signaling is regulated as well as to better strategies for metastasis intervention.
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Acknowledgments |
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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.
We thank Dr. Tadashi Yamamoto for providing the ErbB2 mutant expression vectors; Dr. David Shalloway for providing the Src mutant expression vectors, Drs. Mien-Chie Hung, Jun Yao, and Chris Neal for valuable discussions and comments; and Lore Feldman and Katie Matias for editing the article.
Received 7/ 6/04. Revised 11/ 9/04. Accepted 12/23/04.
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and GTPase-activating protein does not correlate with erbB-2 mitogenic potency. Mol Cell Biol 1991;11:2040–8.. EMBO J 1991;10:2077–86.[Medline]
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C. H. Wong, F. L. Baehner, D. S. Spassov, D. Ahuja, D. Wang, B. Hann, J. Blair, K. Shokat, A. L. Welm, and M. M. Moasser Phosphorylation of the src Epithelial Substrate Trask Is Tightly Regulated in Normal Epithelia but Widespread in Many Human Epithelial Cancers Clin. Cancer Res., April 1, 2009; 15(7): 2311 - 2322. [Abstract] [Full Text] [PDF]
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J. Lu, M. Tan, W.-C. Huang, P. Li, H. Guo, L.-M. Tseng, X.-h. Su, W.-T. Yang, W. Treekitkarnmongkol, M. Andreeff, et al. Mitotic Deregulation by Survivin in ErbB2-Overexpressing Breast Cancer Cells Contributes to Taxol Resistance Clin. Cancer Res., February 15, 2009; 15(4): 1326 - 1334. [Abstract] [Full Text] [PDF]
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 H. Yamaguchi, N. T. Woods, J. F. Dorsey, Y. Takahashi, N. R. Gjertsen, T. Yeatman, J. Wu, and H.-G. Wang Src Directly Phosphorylates Bif-1 and Prevents Its Interaction with Bax and the Initiation of Anoikis J. Biol. Chem., July 4, 2008; 283(27): 19112 - 19118. [Abstract] [Full Text] [PDF]
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 A. Vultur, R. Buettner, C. Kowolik, W. Liang, D. Smith, F. Boschelli, and R. Jove SKI-606 (bosutinib), a novel Src kinase inhibitor, suppresses migration and invasion of human breast cancer cells Mol. Cancer Ther., May 1, 2008; 7(5): 1185 - 1194. [Abstract] [Full Text] [PDF]
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F. Liang, Y. Luo, Y. Dong, C. D. Walls, J. Liang, H.-Y. Jiang, J. R. Sanford, R. C. Wek, and Z.-Y. Zhang Translational Control of C-terminal Src Kinase (Csk) Expression by PRL3 Phosphatase J. Biol. Chem., April 18, 2008; 283(16): 10339 - 10346. [Abstract] [Full Text] [PDF]
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 C. Oneyama, T. Hikita, S. Nada, and M. Okada Functional dissection of transformation by c-Src and v-Src. Genes Cells, January 1, 2008; 13(1): 1 - 12. [Abstract] [Full Text] [PDF]
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S. Yoon, M.-Y. Lee, S. W. Park, J.-S. Moon, Y.-K. Koh, Y.-H. Ahn, B.-W. Park, and K.-S. Kim Up-regulation of Acetyl-CoA Carboxylase {alpha} and Fatty Acid Synthase by Human Epidermal Growth Factor Receptor 2 at the Translational Level in Breast Cancer Cells J. Biol. Chem., September 7, 2007; 282(36): 26122 - 26131. [Abstract] [Full Text] [PDF]
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M. Qiao, J. D. Iglehart, and A. B. Pardee Metastatic Potential of 21T Human Breast Cancer Cells Depends on Akt/Protein Kinase B Activation Cancer Res., June 1, 2007; 67(11): 5293 - 5299. [Abstract] [Full Text] [PDF]
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 R. P. Carnevale, C. J. Proietti, M. Salatino, A. Urtreger, G. Peluffo, D. P. Edwards, V. Boonyaratanakornkit, E. H. Charreau, E. B. de Kier Joffe, R. Schillaci, et al. Progestin Effects on Breast Cancer Cell Proliferation, Proteases Activation, and in Vivo Development of Metastatic Phenotype All Depend on Progesterone Receptor Capacity to Activate Cytoplasmic Signaling Pathways Mol. Endocrinol., June 1, 2007; 21(6): 1335 - 1358. [Abstract] [Full Text] [PDF]
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F. Meric-Bernstam and M.-C. Hung Advances in targeting human epidermal growth factor receptor-2 signaling for cancer therapy. Clin. Cancer Res., November 1, 2006; 12(21): 6326 - 6330. [Abstract] [Full Text] [PDF]
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M. Tan, K.-H. Lan, J. Yao, C.-H. Lu, M. Sun, C. L. Neal, J. Lu, and D. Yu Selective Inhibition of ErbB2-Overexpressing Breast Cancer In vivo by a Novel TAT-Based ErbB2-Targeting Signal Transducers and Activators of Transcription 3-Blocking Peptide. Cancer Res., April 1, 2006; 66(7): 3764 - 3772. [Abstract] [Full Text] [PDF]
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K. S. Klos, S. L. Wyszomierski, M. Sun, M. Tan, X. Zhou, P. Li, W. Yang, G. Yin, W. N. Hittelman, and D. Yu ErbB2 Increases Vascular Endothelial Growth Factor Protein Synthesis via Activation of Mammalian Target of Rapamycin/p70S6K Leading to Increased Angiogenesis and Spontaneous Metastasis of Human Breast Cancer Cells Cancer Res., February 15, 2006; 66(4): 2028 - 2037. [Abstract] [Full Text] [PDF]
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