Skip Navigation


Carcinogenesis Advance Access originally published online on September 6, 2006
Carcinogenesis 2007 28(2):435-445; doi:10.1093/carcin/bgl171
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
28/2/435    most recent
bgl171v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (7)
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Moiseeva, E. P.
Right arrow Articles by Manson, M. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Moiseeva, E. P.
Right arrow Articles by Manson, M. M.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

© The Author 2006. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

EGFR and Src are involved in indole-3-carbinol-induced death and cell cycle arrest of human breast cancer cells

Elena P. Moiseeva*, Raimond Heukers and Margaret M. Manson

Cancer Biomarkers and Prevention Group, Departments of Biochemistry and Cancer Studies University of Leicester, Leicester LE1 7RH, UK

*To whom correspondence should be addressed. Email: em9{at}le.ac.uk


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 Supplementary data
 References
 
Indole-3-carbinol (I3C), a dietary chemopreventive compound, induced marked reduction in epidermal growth factor receptor (EGFR) prior to cell death in cells representing three breast cancer subtypes. Signalling pathways, linking these events were investigated in detail. I3C modulated tyrosine phosphorylation from 30 min in four cell lines. In MDA-MB-468 and HBL100 cells, it induced Src activation after 5 h. In MDA-MB-468 cells, I3C induced signalling between 4.5 and 7 h, which involved sequential activation of Src, EGFR, STAT-1 and STAT-3, followed by EGFR degradation. It also induced physical association between activated Src and EGFR. In MCF7 and MDA-MB-231 cells, I3C modulated expression of cell cycle-related proteins, p21Cip1, p27Kip1, cyclin E, cyclin D1 and CDK6, with upregulation of p21Cip1 and cyclin E being dependent on Src. Inhibition of EGFR by specific inhibitors PD153035 or ZD1839 increased susceptibility to I3C-induced apoptosis of MCF7, MDA-MB-468 and MDA-MB-231 cells. Inhibition of Src sensitized MDA-MB-468 and MDA-MB-231 cells to I3C, whereas overexpression of c-Src increased resistance to I3C in MDA-MB-468 and HBL100 cells. Modulation of Src in MDA-MB-468 cells influenced the basal level of EGFR expression and cell viability; the latter being positively correlated with EGFR activation levels. Therefore, EGFR and Src activities are essential for I3C-induced cell cycle arrest and death; however, I3C-induced pathways depend on specific features of breast cancer cells. The cancer types, which rely on ‘EGFR addiction’ or Src deregulation, are likely to be susceptible to I3C.

Abbreviations: EGFR, epidermal growth factor receptor; ER, estrogen receptor; FAK, focal adhesion kinase; I3C, indole-3-carbinol


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 Supplementary data
 References
 
Many lifestyle-related factors contribute to cancer incidence and epidemiological studies have shown a strong link between cancer risk and diet. Indole-3-carbinol (I3C), derived from cruciferous vegetables, inhibits development of tumours of the skin, tongue, liver, colon, lung, breast and endometrium in animal models [thoroughly reviewed in (1)]. It inhibits cancer development induced by carcinogens and viruses, as well as growth of spontaneous tumours. In phase II trials I3C caused significant regression of cervical intraepithelial neoplasia (CIN) (2) and was a successful treatment option for recurrent respiratory papillomatosis (3). No adverse effects in patients have been reported. Therefore, I3C is regarded as a chemopreventive agent. However, it has been reported to exert a promoting effect when administered during the post-initiation stage of chemical carcinogenesis in rats and trout (1).

I3C exerts several effects that may contribute to cancer prevention. It induces phase I and II enzymes in humans and animal models, leading to increased catabolism and excretion of carcinogens and cancer-promoting steroid hormones (1). A recent phase I trial with women from a high-risk breast cancer cohort proved that I3C is safe and effective, increasing 2-hydroxylation of estradiol (4). Efficacy in CIN treatment involved this mechanism (2). I3C also inhibits cell proliferation and induces apoptosis in tumours in animal models (5–7).

In cell culture, I3C modulates estradiol metabolism and significantly suppresses estrogen receptor (ER{alpha}) signalling via a ligand-dependent mechanism [reviewed in (8)]. It also elicits anti-proliferative and pro-apoptotic responses in cancer cells, including cells refractory to steroid hormones (9–12). Two main molecular mechanisms have been suggested. The first involves modulation of key proteins involved in cell cycle progression, e.g. CDK6, CDK2, p21Waf1/Cip1, p27Kip1, p15INK4B, cyclin D1 and cyclin E (10,11,13–16). Signalling through ATM and p53 has also been implicated in I3C-induced G1 arrest (17). The second mechanism proposes Akt kinase downregulation as a pro-apoptotic event (18–20). However a cause-effect link between Akt downregulation and I3C-induced cell death has not been established in these studies. Moreover, PI3K/Akt downregulation does not account for I3C-induced apoptosis in several cancer lines (12).

We have shown that I3C induced downregulation of epidermal growth factor receptor (EGFR), without affecting pAkt in four breast lines (21), although the decrease in MCF7 cells did not reach statistical significance, possibly because of the low expression level. EGFR downregulation was followed by or coincided with apoptosis via the mitochondrial pathway in MCF7, MDA-MB-468 and MDA-MB-231 cells. We also showed that MDA-MB-468 and MDA-MB-231 cells are EGFR-dependent. In MCF7 cells, I3C-induced apoptosis may be related to combined effects of I3C on ER{alpha} and EGFR. In contrast to cancer-derived cells, I3C does not induce apoptosis, but necrosis in transformed HBL100 cells. I3C-induced EGFR downregulation has been reported in prostate cancer cells PC3 (18). For these reasons we investigated further the role of EGFR in I3C-induced events in breast cancer cells. In MDA-MB-468 cells, EGFR downregulation was preceded by increased phosphorylation of tyrosine EGFR-Y845 and coincided with increased autophosphorylation of EGFR-Y1068 (21). Since phosphorylation of Y845 requires c-Src (22,23), we investigated the role of Src and tyrosine phosphorylation in I3C-induced events. We also examined expression of key proteins involved in cell cycle progression in relation to Src or EGFR activity.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 Supplementary data
 References
 
Cells
Human breast cell lines MCF7, MDA-MB-468, MDA-MB-231, SKBR3 and HBL100 originating from the American Type Culture Collection were cultured as described previously (24). This panel represents cells of four breast cancer subtypes, driven by various deregulated pathways (Supplementary Table).

Cell viability and apoptosis-related studies
Adherent cells were estimated using the ATPlite kit (Perkin–Elmer) as described previously (21). Cells were seeded overnight in DMEM containing 10% FBS, followed by treatments in the same medium for 48 h. Caspase 3/7 activity, as a measure of apoptosis, was determined using a Caspase-Glo 3/7 kit (Promega) as described previously (21). Cells were seeded overnight in 10% FBS in DMEM, followed by treatments in the same medium.

Immunoblotting and immune precipitation
Preparation of cell lysates, SDS–PAGE and immunoblotting were performed as described previously (21). Protein concentrations were measured using the Bio-Rad protein assay. Nitrocellulose filters were incubated with primary antibodies followed by secondary antibody conjugated to horseradish peroxidase (HRP) (Dako) and detected with ECL or ECLplus (EGFR in HBL100 and MCF7 cells) reagent (Amersham). Alternatively secondary antibodies, labelled with fluorescent IR800 and IR680 dyes, were used according to the manufacturer's recommendations and detected with the Odyssey system (Li Cor, UK). Immune complexes were precipitated with protein G-beads or anti-mouse IgG-beads (Sigma) and subjected to western blotting. Protein bands on exposed films were quantified using a Syngene image system.

Antibodies used in this study were against: EGFR (polyclonal sc-1005, Santa Cruz Biotechnology, UK; monoclonal R19/48, polyclonal pY845, pY1148, pY1086 and pY1068, Biosource, UK), Src (monoclonal H-12 c-Src, Santa Cruz Biotechnology, UK, monoclonal 7G9 non-pY416, polyclonal pY416 and pY527, Cell Signaling Technology, UK; polyclonal Src pY418, Biosource, UK), phosphotyrosines (monoclonal PY20, BD Transduction Laboratories, UK; PY99, Santa Cruz Biotechnology, UK, PY100, Cell Signaling Technology), STAT-1 (monoclonal 9H2, Cell Signaling Technology, UK; polyclonal pY701, Biosource, UK), STAT-3 (monoclonal F-2, Santa Cruz Biotechnology, UK; pY705 rabbit monoclonal 58E12, Cell Signaling Technology, UK), ß-actin (polyclonal, Sigma, UK), p21Cip1 (monoclonal SX118, Dako Cytomation, UK), cyclin E (monoclonal HE12, Santa Cruz Biotechnology, UK) and cyclin D1 (monoclonal DSC-6, Dako Cytomation, UK); Her2 (polyclonal 2242, Cell Signaling Technology, UK), ER{alpha} and ERß (monoclonal F-10 and polyclonal sc-8974, Santa Cruz Biotechnology, UK), cytokeratin 5/6 (polyclonal sc-22480, Santa Cruz Biotechnology, UK).

Quantification of gene expression
Total RNA was isolated using a Qiagen kit. cDNA was synthesized at least in duplicate for each sample using a reverse transcription kit (Promega, UK). cDNA transcribed from 50 ng of RNA was used in real-time PCR performed in an ABI 7300 thermocycler to measure the levels of mRNA for caspase-1, p21Cip1, Bcl-xL and 18S RNA using assays-on-demand gene expression kits (Applied Biosystems, UK) with Taqman probes. Levels of expression were calculated using the {Delta}CT method, as described previously (25), with 18S RNA as a reference.

Cell transfection
Cells, plated (5000 cells/well) overnight in 10% FBS in DMEM, were transiently transfected with 100 ng/well of plasmid DNA using 0.2 µl/well of GeneJuice transfection reagent (Novagen, UK) according to the manufacturer's instructions. After 24 h, cells were treated with I3C for another 24 h prior to evaluation of cell number or apoptosis. For protein analysis experiments were scaled up for 6-well plates. A plasmid expressing wild-type chicken Src (pSrc) was kindly provided by Dr Avizienyte (The Beatson Institute for Cancer Research, Glasgow, UK) (26). A pCis-CK plasmid (Stratagene, UK), a negative control vector for cis-reporting signal transduction pathways, was used in parallel. In siRNA experiments, cells were transfected with Src or control siRNA oligonucleotides (Santa Cruz) according to the manufacturer's instructions, as described above; after 24 h cells were treated with I3C for 24 h prior to evaluation of cell number.

Data analysis
Differences among the groups were analyzed using one-way ANOVA, followed by Scheffe's test to determine whether the treatment groups were different or by Dunnett's test to determine whether the treatment groups were different from a control group. The level of statistical significance was selected as P < 0.05. All data are presented as means ± SE.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 Supplementary data
 References
 
Expression of biomarkers of breast cancer sub-types was analyzed in a panel of cell lines (Figure 1A). MCF7 cells expressed several isoforms of ER{alpha} in agreement with previously published data (27). This cell line belongs to the ER{alpha}-positive luminal A sub-type (28,29). All cell lines in the panel expressed ERß. Expression of this receptor was shown previously in our stock of MCF7 and MDA-MB-231 cells (30). Her2 expression was detectable only in SKBR3 cells, previously characterized as ErbB2/Her2 centroid (29). MDA-MB-468, MDA-MB-231 and HBL100 cells did not express either ER{alpha} or Her2 and did not belong to ER{alpha}-positive luminal A/B or Her2 sub-types. MDA-MB-468 and MDA-MB-231 cells belong to the basal-like sub-type, which is characterized by the absence of ER{alpha} and Her2 and expression of EGFR and/or cytokeratin 5/6 (31). The HBL100 cells did not express any of these biomarkers and are likely to be a normal breast-like sub-type. EGFR expression in the four breast lines correlated with reported levels (Supplementary Table), being non-detectable in MCF7 cells in the same conditions as used for other cells (Figure 1A and C, small panels).


Figure 1
View larger version (43K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 1 I3C modulated protein tyrosine phosphorylation. Western blots were developed with the antibodies indicated on the right (A–C) or above the figure (D). Molecular weight markers are indicated. (A) Breast cancer cell lines were analyzed for sub-type biomarker expression. (B) Tyrosine phosphorylation and protein expression in breast tumourigenic cells treated with I3C (µM) for 5 h. The small panel with PY20 antibodies shows a short exposure of a section of the panel above. The positions of EGFR, Src and FAK, shown on small panels, are indicated in italics on the upper panel. (C) Cells were treated with I3C (µM) for 0.5, 1 and 5 h. Molecular weight markers on the right refer to separation of MDA-MB-231 cell extracts; markers on the left refer to HBL100, MCF7 and MDA-MB-468 cells. (D) Extracts from cells treated with I3C (µM) for 5 h were consecutively immune precipitated with anti-FAK antibodies followed by anti-CAS antibodies. Precipitated proteins were separated using SDS–PAGE and detected with PY100 antibody followed by anti-FAK or CAS antibody, respectively.

 
I3C modulated tyrosine phosphorylation
Since I3C induced downregulation of the receptor tyrosine kinase EGFR, we investigated whether I3C affected overall tyrosine phosphorylation. The most I3C-sensitive cells MDA-MB-468 displayed the greatest constitutive levels of tyrosine-phosphorylated proteins, possibly caused by high levels of Src and EGFR (Figure 1B and C, upper panels). Increased tyrosine phosphorylation was first observed in HBL100, MDA-MB-468 and MCF7 cells after 5 h of I3C treatment, while slight decrease was observed in MDA-MB-231 cells (Figure 1B and C). The anti-phospho-tyrosine antibodies PY20 and PY99 produced different phosphorylation patterns due to differences in specificity. The major phospho-tyrosine bands about 170 kDa (MCF7) and 150/170 kDa doublet (MDA-MB-468 cells) were increased ~2-fold and coincided with EGFR in MDA-MB-468 cells. In MDA-MB-468 cells, increased phosphorylation of EGFR-Y845 (190%) appeared greater than that of EGFR-Y1068 (120%) (Figure 1C, small panels). Similar increases were previously reported after 6 h treatment (21). Interestingly, I3C treatment for 0.5–1 h initially decreased phosphorylation at both sites (Figure 1C). No changes in EGFR phosphorylation were observed in MDA-MB-231 cells (Figure 1C, lower panels). No phosphorylated EGFR was detectable in HBL100 or MCF7 cells (data not shown).

In MDA-MB-468 cells, probed with PY20, a protein of about 120 kDa [suggestive of focal adhesion kinase (FAK) or p130CAS, both of which are Src substrates] showed a 5-fold increase in phospho-tyrosine following treatment with 125 µM I3C (Figure 1B, C and data not shown). A 4-fold increase in phosphorylation of a similar band was detected in I3C-induced HBL100 cells with PY99, but not PY20 antibody (compare Figure 1B and C). To identify the phosphorylated protein, we performed a sequential immune precipitation with anti-FAK antibodies, followed by anti-CAS antibodies (Figure 1D). FAK was slightly phosphorylated only in MDA-MB-468, whereas p130CAS was significantly tyrosine-phosphorylated in both I3C-treated cell lines, indicating that the 120 kDa band is more likely to be p130CAS.

Since EGFR-Y845 and p130CAS are Src substrates, we investigated whether Src was affected by I3C treatment using antibodies against activated Src (autophosphorylated on Y418), non-activated Src (non-phospho-Y418 Src) and non-activated Src in closed conformation (Src-pY529). Increased phosphorylation of Src-Y418 was observed after 5 h treatment in HBL100 (up to 350%) and MDA-MB-468 (190%) cells (Figure 1C). No increase was observed in MCF7 cells and phosphorylation of Src-pY418 gradually decreased to 18% after 5 h in the MDA-MB-231 cells (Figure 1C). Taken together, these data show that I3C modulated tyrosine phosphorylation in all four breast lines.

Effect of I3C on EGFR and Src signalling in MDA-MB-468 cells
We next analysed the sequence of events in MDA-MB-468 cells in response to I3C. Significant increase in phosphorylation of Src-Y418 occurred between 4.5–5.5 h, reaching a 6-fold maximum at 5 h (Figure 2A). No co-incident decrease in inactive Src (non-phosphorylated Y418 or Src-pY529) was observed (Figure 2A), which implied that only a small proportion became activated or activation occurred via pY529-independent mechanism (32). Activated c-Src phosphorylates EGFR on Y845 in this cell line (22). I3C-induced phosphorylation of EGFR-Y845 was detected from 5 h, followed by increased autophosphorylation of Y1068 and Y1086 from 5.5 h (Figure 2A). The increases in tyrosine phosphorylation were preceded by a noticeable decrease at all three EGFR-Y sites from 3.5 to 5 h (Figure 2A), as reported previously (21). These events were followed by EGFR degradation from 5.5 h (Figure 2A), again corresponding to our previously reported data (21). Phosphorylated EGFR is degraded following ubiquitination (33). In MDA-MB-468 cells, both Src and EGFR are involved in activation of STATs 1, 3 and 5, resulting in dimerization and nuclear translocation of these transcription factors (34,35). Increased phosphorylation of STAT-1-Y701 (preceded by a decrease) and STAT-3-Y705 was observed from 5.5 h, both increasing to 150% by 7 h (Figure 2A). An insignificant decrease in phosphorylation of Akt-S473 was detected from 7 h (Figure 2A). Altogether, these data indicated that I3C activated Src, which initiated a sequence of signalling involving EGFR, STAT-1 and STAT-3 in MDA-MB-468 cells.


Figure 2
View larger version (78K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 2 I3C-induced signalling in MDA-MB-468 cells involved Src and EGFR. (A) MDA-MB-468 cells were treated with 125 µM I3C for 3.5–7 h. Western blots were developed with antibodies shown. (B and C) MDA-MB-468 cells were treated with 125 µM I3C and 10 µM PP2 (C) for 5 h. Western blots were developed with the antibodies indicated. Proteins were analyzed in whole cell extract (C) or immune-precipitated with either anti-c-Src or anti-EGFR antibodies (B).

 
It has been reported that Src binds to activated EGFR in MDA-MB-468 cells (36). Since I3C induced tyrosine phosphorylation of both Src and EGFR, we investigated if they became physically associated in response to such treatment. Indeed, EGFR was bound to immune-precipitated c-Src, and conversely activated Src-pY418 was co-precipitated with EGFR in I3C-stimulated cells (Figure 2B). I3C-induced phosphorylation of Src-Y418 and Src-Y529 was significantly inhibited by the specific Src inhibitor PP2 (Figure 2C), both are autophosphorylation sites (37,38). PP2 also reduced phosphorylation of EGFR (on Src-phosphorylated Y-845 and autophosphorylated sites Y1086, Y1068 and Y1148) and STAT-1 (Figure 2C). Phosphorylation of Akt or STAT-3 was not affected by PP2.

Effect of Src and EGFR on I3C-induced modulation of cell cycle-related proteins
We previously showed that I3C reduces EGFR levels to 30 ± 12, 44 ± 22 and 31 ± 8% at 30 h treatment in HBL100, MCF7 and MDA-MB-231 cells, respectively, while expression in MDA-MB-468 had returned to 86 ± 9% by this time (21) (Figure 3A and B). I3C has been reported to modulate p21Cip1, p27Kip1 and CDK6 in MCF7 cells and induced cell cycle arrest in MCF7 and MDA-MB-231 cells (10). Here we investigated whether five proteins involved in cell cycle were modulated by I3C following EGFR downregulation. No such changes were found in HBL100 or MDA-MB-468 cells. Expression of p21Cip1 protein was increased in MCF7 and, particularly, in MDA-MB-231 cells after 24 h and cyclin D1 was reduced in MCF7 and MDA-MB-231 cells at 30 h (Figure 3A and B). Cyclin E was increased by I3C particularly in MCF7 cells, but the increase did not reach statistical significance (P = 0.075). Preliminary experiments in MDA-MB-231 cells indicated that expression of p27Kip1 and CKD6 was also modulated by I3C (Figure 3A). The data on expression of p21Cip1, cyclin E and cyclin D1 in I3C-treated MCF7 cells are very similar to those, previously published, although in that study changes in cyclin E and cyclin D1 were not considered significant, when monitored in 24 h intervals (10). Since p21Cip1 expression can be regulated at a post-transcriptional level (39) and it is induced by I3C at a transcriptional level (11), we examined mRNA in MDA-MB-468 cells by real-time PCR. Bcl-xL and caspase-1 were also examined, since they can also be modulated by I3C at protein and RNA levels (18,19,40). We did not detect any significant change in caspase-1 or Bcl-xL gene expression (Figure 3C). However, p21Cip1 mRNA was significantly upregulated by I3C in MDA-MB-468 cells starting from 3 h of treatment, despite no apparent increase in protein levels (Figure 3A).


Figure 3
View larger version (39K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 3 I3C induced modulation of expression of cell cycle-related proteins. (A) Protein expression in HBL100, MCF7, MDA-MB-468 and MDA-MB-231 cells, treated with I3C for 18–30 h. (B) Quantified results of three experiments (A) are shown. EGFR (A and B) expression is reproduced from Figure 7 published in (21) with kind permission of Springer Science and Business Media. (C) Total RNA was isolated from MDA-MB-468 cells treated with 125 µM I3C for 0, 3, 15 and 21 h. The levels of mRNA for caspase-1, p21Cip1 and Bcl-xL were measured. (n = 6) *, P < 0.05 versus ‘no I3C’.

 
We next investigated whether Src or EGFR kinase activities are necessary for I3C-related modulation of cell cycle proteins and EGFR (Figure 4). Treatment with Src and EGFR specific inhibitors PP2 and PD153035, respectively, individually did not affect expression of proteins investigated, with the exception of p27Kip1, which was decreased to 21 ± 7% (P < 0.05) by EGFR inhibition in MDA-MB-231 cells, and cyclin D1, which was decreased to 54 ± 8 and 58 ± 10% (P < 0.05) by Src or EGFR inhibition, respectively, in MCF7 cells (Figure 4A and B). Regulation of cyclin D1 by EGFR in MCF7 cells is consistent with the published data (41). In the presence of I3C expression of p21Cip1, cyclin E and p27Kip1 was increased, whereas expression of EGFR, cyclin D1 and CDK6 was decreased in both MCF7 and MDA-MB-231 cells (Figure 4A and B). Increase in cyclin E was small, particularly in MDA-MB-231 cells, but here reached statistical significance. When I3C was combined with each inhibitor, EGFR downregulation was not prevented by inhibition of either Src or EGFR activities in either cell line (Figure 4A and B). I3C-induced upregulation of p21Cip1 and cyclin E was abrogated by Src inhibition in MCF7 and MDA-MB-231 cells (Figure 4A and B). In MDA-MB-468 cells, the I3C-induced increase in p21Cip1 mRNA was reduced by Src inhibition (Figure 4C). I3C-induced reduction of cyclin D1 levels in MCF7 cells is likely to be a result of EGFR downregulation. The data are summarized in Figure 8.


Figure 4
View larger version (60K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 4 I3C-induced upregulation of p21Cip1 and cyclin E involved Src. (A) Protein expression in MCF7 and MDA-MB-231 cells, pre-treated with 10 µM PP2, or 100 nM PD153035 for 40 min prior to treatment with 250 µM I3C for 30 h. (B) Quantified results of three experiments (A) are shown. The data for treatments with inhibitors are shown only for those proteins, for which expression was changed by the treatments. (C) MDA-MB-468 cells were induced by 125 µM I3C with/without 10 µM PP2 for 8 h. The levels of p21Cip1 mRNA were measured (n = 6). *, P < 0.05 versus ‘no I3C’. #, P < 0.05 versus ‘no inhibitor’.

 
Modulation of EGFR and Src influenced susceptibility to I3C
Since I3C-induced signalling involved Src and EGFR, we examined whether altered activity of these proteins could influence susceptibility to I3C. In MDA-MB-468 cells, the EGFR inhibitor PD153035 (50 nM) did not change the autophosphorylation status (Y1068) of EGFR, but increased the amounts of non-active Src (non-pY418) up to 181% (Figure 5A). PP2 reduced Src activation (phosphorylation of Y418) to 40% and increased amounts of non-phosphorylated Src-Y418 to 260% (Figure 5A). Inhibition of Src reduced EGFR autophosphorylation on Y1068 by 20% and markedly increased total EGFR up to 386% (Figure 5A), probably due to reduced degradation of hypo-phosphorylated EGFR (33). These data, as well as data in Figure 2, indicated that Src and EGFR are inter-dependent in MDA-MB-468 cells. Pharmacological inhibition of Src and/or EGFR increased apoptosis and reduced cell viability in I3C-treated MDA-MB-468 cells (Figure 5A). Downregulation of Src by siRNA (50 nM) decreased amounts of total c-Src protein by half, as well as its activated form Src-pY418 and phosphorylation of EGFR-pY845 (Figure 5B). These changes also resulted in reduced viability of I3C-treated MDA-MB-468 cells (Figure 5B). Thus, downregulation of Src by siRNA or pharmacologically led to reduction in activated EGFR and increased sensitivity to I3C in MDA-MB-468 cells. Similarly, inhibition of Src or EGFR resulted in increased apoptosis in I3C-treated MDA-MB-231 cells, but did not alter viability of the other two cell lines (Figure 5C). Because MCF7 cells lack functional caspase-3 and HBL100 cells undergo necrosis in response to I3C (21), cell viability assays were used.


Figure 5
View larger version (39K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 5 Downregulation of Src and/or EGFR increased sensitivity of MDA-MB-468 and MDA-MB-231 cells to I3C. (A) MDA-MB-468 cells were treated with 125 µM I3C (if indicated) in combination 10 µM PP2, 50 or 100 nM (if indicated) PD153035 for 24 h. Protein expression, cell viability (n = 24) and apoptosis (n = 12) were examined. *, P < 0.05 versus ‘no I3C’. #, P < 0.05 compared to ‘no inhibitor’ (n = 24). (B) Expression of Src and EGFR in cells transfected with indicated concentrations of Src siRNA for 24 h. Viability of cell transfected with 40 nM of control or Src siRNAs followed by 24 h treatment with 125 µM I3C. *, P < 0.05 versus ‘no I3C’; #, P < 0.05 versus ‘control’ (n = 12). (C) Viability or apoptosis in cells treated with 250 µM I3C in combination with 10 µM PP2 or 100 nM PD153035 for 24 h. *, P < 0.05 versus ‘no I3C’; #, P < 0.05 compared to ‘no inhibitor’ (n = 12).

 
Since I3C susceptibility was enhanced by EGFR downregulation, we investigated the efficacy of combined treatment with I3C and ZD1839 (Iressa), an EGFR-specific inhibitor used in the treatment of lung cancer patients and in trials with breast cancer patients. Although, both MDA-MB-468 and MDA-MB-231 cells respond to ZD1839, only the former are sensitive to 1 µM (Figure 6) in good agreement with published data on ZD1839 susceptibility of these cells (42,43). Combined I3C+ZD1839 treatment increased caspase 3/7 activity in MDA-MB-468 and MDA-MB-231 cells and decreased viability in these and in MCF7 cells (Figure 6). Hence, the combinational treatment increased death not only in MDA-MB-468 and MDA-MB-231 cells, but also in MCF7 cells, which are not sensitive to ZD1839 alone (Figure 6).


Figure 6
View larger version (32K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 6 ZD1839 increased susceptibility to I3C. (A) Apoptosis in cells treated with indicated concentrations of I3C (µM) in combination with 1 µM ZD1839 for 30 h. (B) Viability of cells, treated as in A for 48 h. *, P < 0.05 versus ‘no I3C’; #, P < 0.05 compared to ‘no inhibitor’ (n = 8).

 
Finally, to confirm the involvement of Src in I3C action, we used transient overexpression of chicken wild-type Src. Since the antibody against c-Src did not recognize chicken Src, we used antibodies against Src-pY418, non-phosphorylated Src-Y418 and Src-pY529, which recognized both human and chicken Src. Overexpression of wild-type Src in MDA-MB-468 cells increased the levels of autophosphorylation of Y418 by 6-fold and non-pY418 by 2.3-fold, whereas Src-pY529 increased to 142%. Overexpression of Src also increased phosphorylation of EGFR-Y1068 by 3.2-fold and total EGFR amount by 1.6-fold compared to cells transfected with a control vector (Figure 7A). Similar upregulation of EGFR is reported for other transfected cell lines, overexpessing Src, due to increased c-Cbl degradation (44). Transient overexpression of wild-type Src reduced susceptibility of MDA-MB-468 cells to I3C (Figure 7A). Similarly, overexpression of wild-type Src abrogated I3C-induced cell death in HBL100, but not in MDA-MB-231 or MCF7 cells (Figure 7B). Hence, overexpression of Src increased I3C resistance in two breast tumour cell lines. The data are summarized in Figure 8.


Figure 7
View larger version (37K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 7 Overexpression of Src increased resistance to I3C in MDA-MB-468 and HBL100 cells. Cell viability and apoptosis were measured in cells transiently transfected with expression pSrc plasmid or control pCK for 24 h and treated with indicated concentrations of I3C for another 24 h. (A) Data for MDA-MB-468 cells. Western blot analysis of expression of Src and EGFR after 24 h transfection. (B) Data for HBL100, MCF7 and MDA-MB-231 cells. *, P < 0.05 compared with ‘no I3C’; #, P < 0.05 compared to cells transfected with pSrc (n = 16).

 


Figure 8
View larger version (25K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 8 A diagram summarizes the molecular mechanisms of I3C-induced cell cycle arrest and apoptosis. The diagram shows that I3C induces apoptosis via downregulation of ER{alpha} and EGFR in ER-positive cancer cells or via EGFR in ER-negative cells, viability of which is regulated by EGFR ligands. Cells, such as HBL100, do not undergo apoptosis in response to I3C. Induction of p21Cip1 and cyclin E during cell cycle arrest requires Src activity, whereas downregulation of cyclin D1 requires downregulation of EGFR in some cancer cells. Upregulation of p21Cip1 mRNA does not necessarily lead to increased protein levels, shown as (468). The numbers in the grey boxes indicates the cell lines. Signalling pathways based on published data are shown in grey.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
Supplementary data
 References
 
I3C modulated tyrosine phosphorylation
The data presented here indicate that I3C modulated tyrosine phosphorylation as an early event in all cell lines, although the pattern varied in different cell lines. Phosphorylation of EGFR and, possibly, p130CAS were the most striking events in MDA-MB-468 cells and HBL100 cells, as detected by pan-phospho-tyrosine antibodies. Changes in phosphorylation of other proteins were detected only by specific antibodies. Detailed examination of I3C-induced signalling in MDA-MB-468 cells revealed that dephosphorylation of EGFR and STAT1 was followed by activation of Src, which led to EGFR activation, followed by activation of STAT-1, STAT-3 and Erk (21). Src-mediated signalling culminated in EGFR downregulation and the onset of apoptosis. EGFR downregulation was observed in all cell types, whereas other changes were observed only in some of them. Activation of Src and phosphorylation of p130 kDa was found in HBL100 cells. Phosphorylation of protein similar in molecular weight to EGFR was detected in MCF7 cells. Tyrosine dephosphorylation was detected in MDA-MB-231 cells. Elements of this signalling have been reported in other models. EGFR dephosphorylation prior to activation has been observed (21). I3C was shown to decrease tyrosine phosphorylation in the liver of rats while inhibiting the development of pre-neoplastic lesions in a carcinogen-induced hepatocarcinogenesis model (45). STAT-1 has been implicated in a combined response to I3C and interferon in MCF7 cells (46).

The role of EGFR in I3C-induced cell death
Several lines of evidence indicate that EGFR downregulation is responsible for I3C-induced apoptosis. First, it is the only common event so far detected in I3C-treated cells (21). Second, the timescale of I3C-induced reduction in EGFR levels in breast cancer cell lines was inversely related to susceptibility, i.e. after 6 h in the most I3C-susceptible MDA-MB-468 cells and after 24–30 h in other cells (21). Third, EGFR inhibitors PD153035 or ZD1839 increased I3C-induced cell death in all breast cancer cell lines, but not HBL100. In contrast, to other breast cancer cells or normal fibroblasts, the ER-negative HBL100 cells are entirely insensitive to downregulation of EGFR and do not undergo apoptosis in the presence of I3C (21). Published data indicate that these cells require bFGF, which they secrete, whereas the other cells require EGFR ligands for growth (Supplementary Table). The HBL100 cells can be sensitized to I3C by blocking protein synthesis during treatment (21), which would inhibit FGF synthesis. It is likely that FGF signalling overrides EGFR signalling in these cells. Finally, downregulation of activated EGFR by inhibition of Src, pharmacologically or by siRNA, increased cell death in MDA-MB-468, while upregulation of EGFR by overexpression of Src decreased susceptibility to I3C.

ER{alpha}-negative MDA-MB-468 and MDA-MB-231 cells are ‘EGFR-dependent’ with MDA-MB-468 cells the most sensitive to downregulation (21). They undergo apoptosis in the presence of 1 µM ZD1839, whereas MDA-MB-231 cells are not susceptible to this concentration (Figure 6). The effect of EGFR inhibition on viability of ER{alpha}-positive MCF7 cells is small (21). Neither I3C (50 and 100 µM) nor 1 µM ZD1839 inhibited these cells; however, in combination, they reduced viability. At higher doses, I3C-induced apoptosis in MFC7 cells is likely to be caused by combined I3C effect on ER{alpha} and EGFR.

The role of Src-EGFR interactions in response to I3C
Src became physically associated with activated EGFR in I3C-treated MDA-MB-468 cells and modulation of either affected the other partner in the complex (Figures 2C, 5A, B and 7A). In untreated MDA-MB-231 cells, EGFR-Y845 phosphorylation was not detectable, even though Src was active and EGFR-Y1068 was markedly phosphorylated (Figure 1C). Src–EGFR interactions are less significant in cells with low receptor expression (36). Besides transactivation, Src is involved in other aspects of EGFR signalling by regulating the basal levels of EGFR via internalization and degradation via phosphorylation of dynamin, clathrin and Cbl (47). In agreement with this, Src modulated basal EGFR levels, as well as activation (Figures 5A and 7A). Src also couples EGFR to G-protein-coupled receptors and adhesion receptors, integrins (47). This may explain why I3C responses are modulated in 3D culture, where cell adhesion is modified compared to monolayer culture (21). Moreover, Src may also mediate some I3C responses via p130CAS in MDA-MB-468 and HBL100 cells. Furthermore, ER{alpha} signalling involves Src and its downstream transducer p130CAS (47,48). It is interesting that both EGFR and Src are implicated in resistance to chemotherapeutic treatments (42,49). Downregulation of Src increased I3C susceptibility in MDA-MB-468 and MDA-MB-231 cells, whereas Src overexpression caused I3C resistance in MDA-MB-468 and HBL100 cells. Importantly, breast cancer cells with Src deregulated pathways, such as MDA-MB-231 cells (50), are likely to be more susceptible to combined I3C+Src inhibition treatments.

Roles of Src and EGFR in I3C-induced modulation of cell cycle-related proteins
I3C has been reported to modulate cell cycle-related proteins, p21Cip1, p27Kip1 and CDK6, in MCF7 cells and to induce cell cycle arrest in MCF7 and MDA-MB-231 cells (10). Moreover, it activates transcription of p21Cip1 in prostate cancer PC3 cells (11). Here I3C induced upregulation of p21Cip1, p27Kip1 and cyclin E and downregulation of CDK6 and cyclin D1 not only in MCF7, but also in MDA-MB-231. Recently I3C-induced p21Cip1 upregulation has been related to p53 function (17) in contradiction to I3C-induced p21Cip1 increase in p53 mutant or p53-null cells (11,17). Our data also indicate that p21Cip1 protein or mRNA upregulation occurred in p53-mutant MDA-MB-231 and MDA-MB-468 cells, respectively (Supplementary Table). Interestingly, p21Cip1 was upregulated in a Src-dependent manner at mRNA, but not protein level in MDA-MB-468 cells. These cells are delayed in S-phase by I3C treatment (M. Manson and L. Fox, unpublished data), in contrast to other cell lines delayed in G1 phase (10,13). We investigated involvement of Src and EGFR in I3C-induced modulation of these cell cycle-related proteins. Src activity was essential for I3C-induced increases in the levels of p21Cip1 and cyclin E in MCF7 and MDA-MB-231 cells and p21Cip1 mRNA increase in MDA-MB-468 cells. Src involvement in cyclin E upregulation has been reported (51). It is possible that simultaneous upregulation of p21Cip1 and cyclin E is essential to I3C action, generating a cytoplasmic inactive p21Cip1–cyclin E-CDK2 75 kDa complex (16). I3C-induced downregulation of cyclin D1 levels in MCF7 cells is likely to be a result of EGFR downregulation, since EGFR inhibition by PD153035 also resulted in decreased cyclin D1 in these cells. EGFR signalling and nuclear EGFR in particular have been implicated in cyclin D1 expression (41,52). Thus, both Src and EGFR are involved in I3C-induced cell cycle arrest.

Importantly I3C-induced modulation of cell cycle-related proteins occurred after 24–30 h treatment, whereas loss of mitochondrial potential was initiated by 16 h treatment in MCF7 cells and phosphatidylserine externalization was detected in MDA-MB-231 cells after 24 h treatment (21). Therefore, the onset of apoptosis preceded or coincided with the cell cycle arrest-related changes. p21Cip1 is implicated in apoptosis, as well as in cell cycle arrest (39). In agreement with this, p21Cip1 induction was reported during apoptosis induced by EGFR downregulation by ZD1839 in MDA-MB-231 cells (43). Downregulation by PD153035 did not lead to increased p21Cip1 in MDA-MB-231 cells in this study, possibly, because we used a much less potent dose to avoid apoptotic events. In MDA-MB-468 cells, EGFR activation by EGF results in apoptosis (53), which is mediated by STAT-1 and involves upregulation of p21Cip1 and caspase activity (54,55). We found some differences between apoptosis induced by I3C and EGF. The upregulation of p21Cip1 mRNA occurred before activation of STAT-1 (compare Figures 2A and 3C).

In conclusion, the data presented here implicate Src, EGFR and tyrosine phosphorylation in the I3C-induced response in three breast cancer subtypes. However, further investigations are required to determine the primary target of I3C action. Importantly, our data show for the first time that involvement of EGFR and Src is essential to I3C action. They indicate that the apoptosis and cell cycle arrest observed after I3C treatment share some common signalling pathways related to Src and/or EGFR activity, and the impact on cancer cells is determined by specific characteristics of the cancer type. Nonetheless, cells representing luminal A (MCF7), basal-like (MDA-MB-468 and MDA-MB-231) and possibly normal breast-like (HBL100) subtypes of breast cancer are responsive to I3C. Src and EGFR signalling may not be coupled upon I3C treatment in every cancer cell type; however, we would speculate that I3C could affect the majority of EGFR- and Src-dependent cancer types via its effect on one or other of these important signal transduction mediators. Deregulation of the Src pathway is found in several clusters of breast cancer with different prognosis (50). EGFR is overexpressed in a significant proportion of breast cancer (49,56), particularly in the basal subtype (31), and distinct alterations in the CA repeat of the EGFR gene are prognostic in breast cancer (57). Therefore, a significant group of breast cancers with deregulated Src and EGFR pathways is likely to be sensitive to I3C. Translational studies are required to examine efficacy of I3C treatment in various subtypes of breast cancer, particularly in combination with clinical Src- or EGFR-specific inhibitors.


   Supplementary data
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary data
References
 
Supplementary data are available at Carcinogenesis Online.


   Acknowledgments
 
The authors are grateful to Dr Avizienyte (The Beatson Institute for Cancer Research, Glasgow, UK) for her kind gift of the Src-expressing plasmid and to AstraZeneca for providing ZD1839. The authors are grateful to L.A.F. Temple for assistance with some data in Figure 7. This work was funded by the UK Medical Research Council Grant No.G0100872.

Conflict of Interest Statement: None declared.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary data
 References
 

  1. IARC Working Group on the Evaluation of Cancer Preventive Strategies. (2004) Cruciferous Vegetables, Isothiocyanates and Indoles. IARC Handbooks on Cancer Prevention(IARC Press, Lyon) Vol. 9: pp. 1–246.
  2. Bell M.C., Crowley-Nowick P., Bradlow H.L., Sepkovic D.W., Schmidt-Grimminger D., Howell P., Mayeaux E.J., Tucker A., Turbat-Herrera E.A., Mathis J.M. (2000) Placebo-controlled trial of indole-3-carbinol in the treatment of CIN. Gynecol. Oncol. 78:123–129.[CrossRef][ISI][Medline]
  3. Rosen C.A. and Bryson P.C. (2004) Indole-3-carbinol for recurrent respiratory papillomatosis: long-term results. J. Voice 18:248–253.[CrossRef][ISI][Medline]
  4. Reed G.A., Peterson K.S., Smith H.J., Gray J.C., Sullivan D.K., Mayo M.S., Crowell J.A., Hurwitz A. (2005) A phase I study of indole-3-carbinol in women: tolerability and effects. Cancer Epidemiol. Biomarkers Prev. 14:1953–1960.[Abstract/Free Full Text]
  5. Chen D.Z., Qi M., Auborn K.J., Carter T.H. (2001) Indole-3-carbinol and diindolylmethane induce apoptosis of human cervical cancer cells and in murine HPV16-transgenic preneoplastic cervical epithelium. J. Nutr. 131:3294–3302.[Abstract/Free Full Text]
  6. Zhang X. and Malejka-Giganti D. (2003) Effects of treatment of rats with indole-3-carbinol on apoptosis in the mammary gland and mammary adenocarcinomas. Anticancer Res. 23:2473–2479.[ISI][Medline]
  7. Katdare M., Singhal H., Newmark H., Osborne M.P., Telang N.T. (1997) Prevention of mammary preneoplastic transformation by naturally-occurring tumor inhibitors. Cancer Lett. 111:141–147.[CrossRef][ISI][Medline]
  8. Kim Y.S. and Milner J.A. (2005) Targets for indole-3-carbinol in cancer prevention. J. Nutr. Biochem. 16:65–73.[CrossRef][ISI][Medline]
  9. Telang N.T., Katdare M., Bradlow H.L., Osborne M.P., Fishman J. (1997) Inhibition of proliferation and modulation of estradiol metabolism: novel mechanisms for breast cancer prevention by the phytochemical indole-3-carbinol. Proc. Soc. Exp. Biol. Med. 216:246–252.[Abstract]
  10. Cover C.M., Hsieh S.J., Tran S.H., Hallden G., Kim G.S., Bjeldanes L.F., Firestone G.L. (1998) Indole-3-carbinol inhibits the expression of cyclin-dependent kinase-6 and induces a G1 cell cycle arrest of human breast cancer cells independent of estrogen receptor signaling. J. Biol. Chem. 273:3838–3847.[Abstract/Free Full Text]
  11. Chinni S.R., Li Y., Upadhyay S., Koppolu P.K., Sarkar F.H. (2001) Indole-3-carbinol (I3C) induced cell growth inhibition, G1 cell cycle arrest and apoptosis in prostate cancer cells. Oncogene 20:2927–2936.[CrossRef][ISI][Medline]
  12. Howells L.M., Hudson E.A., Manson M.M. (2005) Inhibition of phosphatidylinositol 3-kinase/protein kinase B signaling is not sufficient to account for indole-3-carbinol-induced apoptosis in some breast and prostate tumor cells. Clin. Cancer Res. 11:8521–8527.[Abstract/Free Full Text]
  13. Cram E.J., Liu B.D., Bjeldanes L.F., Firestone G.L. (2001) Indole-3-carbinol inhibits CDK6 expression in human MCF-7 breast cancer cells by disrupting Sp1 transcription factor interactions with a composite element in the CDK6 gene promoter. J. Biol. Chem. 276:22332–22340.[Abstract/Free Full Text]
  14. Zhang J., Hsu B.A.J., Kinseth B.A.M., Bjeldanes L.F., Firestone G.L. (2003) Indole-3-carbinol induces a G1 cell cycle arrest and inhibits prostate-specific antigen production in human LNCaP prostate carcinoma cells. Cancer 98:2511–2520.[CrossRef][ISI][Medline]
  15. Matsuzaki Y., Koyama M., Hitomi T., Kawanaka M., Sakai T. (2004) Indole-3-carbinol activates the cyclin-dependent kinase inhibitor p15(INK4b) gene. FEBS Lett. 576:137–140.[CrossRef][ISI][Medline]
  16. Garcia H.H., Brar G.A., Nguyen D.H., Bjeldanes L.F., Firestone G.L. (2004) Indole-3-carbinol (I3C) inhibits cyclin dependent kinase-2 function in human breast cancer cells by regulating the size distribution, associated cyclin E forms and subcellular localization of the CDK2 protein complex. J. Biol. Chem. 280:8756–8764.
  17. Brew C.T., Aronchik I., Hsu J.C., Sheen J.H., Dickson R.B., Bjeldanes L.F., Firestone G.L. (2006) Indole-3-carbinol activates the ATM signaling pathway independent of DNA damage to stabilize p53 and induce G1 arrest of human mammary epithelial cells. Int. J. Cancer 118:857–868.[CrossRef][ISI][Medline]
  18. Chinni S.R. and Sarkar F.H. (2002) Akt inactivation is a key event in indole-3-carbinol-induced apoptosis in PC-3 cells. Clin. Cancer Res. 8:1228–1236.[Abstract/Free Full Text]
  19. Howells L.M., Gallacher-Horley B., Houghton C.E., Manson M.M., Hudson E.A. (2002) Indole-3-carbinol inhibits protein kinase B/Akt and induces apoptosis in the human breast tumor cell line MDA MB468 but not in the nontumorigenic HBL100 line. Mol. Cancer Ther. 1:1161–1172.[Abstract/Free Full Text]
  20. Rahman K.M., Li Y., Sarkar F.H. (2004) Inactivation of akt and NF-kappaB play important roles during indole-3-carbinol-induced apoptosis in breast cancer cells. Nutr. Cancer 48:84–94.[CrossRef][ISI][Medline]
  21. Moiseeva E.P., Fox L.H., Howells L.M., Temple L.A.F., Manson M.M. (2006) Indole-3-carbinol-induced death in cancer cells involves EGFR downregulation and is exacerbated in a 3D environment. Apoptosis 11:799–812.[CrossRef][ISI][Medline]
  22. Biscardi J.S., Maa M.C., Tice D.A., Cox M.E., Leu T.H., Parsons S.J. (1999) c-Src-mediated phosphorylation of the epidermal growth factor receptor on Tyr845 and Tyr1101 is associated with modulation of receptor function. J. Biol. Chem. 274:8335–8343.[Abstract/Free Full Text]
  23. Sato K., Nagao T., Iwasaki T., Nishihira Y., Fukami Y. (2003) Src-dependent phosphorylation of the EGF receptor Tyr-845 mediates Stat-p21waf1 pathway in A431 cells. Genes Cells 8:995–1003.[Abstract]
  24. Gordon L.A., Mulligan K.T., Maxwell-Jones H., Adams M., Walker R.A., Jones J.L. (2003) Breast cell invasive potential relates to the myoepithelial phenotype. Int. J. Cancer 106:8–16.[CrossRef][ISI][Medline]
  25. Fang M.Z., Chen D., Sun Y., Jin Z., Christman J.K., Yang C.S. (2005) Reversal of hypermethylation and reactivation of p16INK4a, RARbeta, and MGMT genes by genistein and other isoflavones from soy. Clin. Cancer Res. 11:7033–7041.[Abstract/Free Full Text]
  26. Avizienyte E., Wyke A.W., Jones R.J., McLean G.W., Westhoff M.A., Brunton V.G., Frame M.C. (2002) Src-induced de-regulation of E-cadherin in colon cancer cells requires integrin signalling. Nat. Cell. Biol. 4:632–638.[ISI][Medline]
  27. Fasco M.J., Keyomarsi K., Arcaro K.F., Gierthy J.F. (2000) Expression of an estrogen receptor alpha variant protein in cell lines and tumors. Mol. Cell. Endocrinol. 166:156–169.[Medline]
  28. Sorlie T. (2004) Molecular portraits of breast cancer: tumour subtypes as distinct disease entities. Eur. J. Cancer 40:2667–2675.[CrossRef][ISI][Medline]
  29. Bertucci F., Finetti P., Rougemont J., et al. (2005) Gene expression profiling identifies molecular subtypes of inflammatory breast cancer. Cancer Res. 65:2170–2178.[Abstract/Free Full Text]
  30. Shaw J.A., Udokang K., Mosquera J.M., Chauhan H., Jones J.L., Walker R.A. (2002) Oestrogen receptors alpha and beta differ in normal human breast and breast carcinomas. J. Pathol. 198:450–457.[CrossRef][ISI][Medline]
  31. Nielsen T.O., Hsu F.D., Jensen K., et al. (2004) Immunohistochemical and clinical characterization of the basal-like subtype of invasive breast carcinoma. Clin. Cancer Res. 10:5367–5374.[Abstract/Free Full Text]
  32. Akhand A.A., Pu M., Senga T., Kato M., Suzuki H., Miyata T., Hamaguchi M., Nakashima I. (1999) Nitric oxide controls src kinase activity through a sulfhydryl group modification-mediated Tyr-527-independent and Tyr-416-linked mechanism. J. Biol. Chem. 274:25821–25826.[Abstract/Free Full Text]
  33. Ettenberg S.A., Rubinstein Y.R., Banerjee P., Nau M.M., Keane M.M., Lipkowitz S. (1999) cbl-b inhibits EGF-receptor-induced apoptosis by enhancing ubiquitination and degradation of activated receptors. Mol. Cell. Biol. Res. Commun. 2:111–118.[CrossRef][Medline]
  34. Garcia R., Bowman T.L., Niu G., et al. (2001) Constitutive activation of Stat3 by the Src and JAK tyrosine kinases participates in growth regulation of human breast carcinoma cells. Oncogene 20:2499–2513.[CrossRef][ISI][Medline]
  35. Kloth M.T., Laughlin K.K., Biscardi J.S., Boerner J.L., Parsons S.J., Silva C.M. (2003) STAT5b, a mediator of synergism between c-Src and the epidermal growth factor receptor. J. Biol. Chem. 278:1671–1679.[Abstract/Free Full Text]
  36. Biscardi J.S., Belsches A.P., Parsons S.J. (1998) Characterization of human epidermal growth factor receptor and c-Src interactions in human breast tumor cells. Mol. Carcinog. 21:261–272.[CrossRef][ISI][Medline]
  37. Osusky M., Taylor S.J., Shalloway D. (1995) Autophosphorylation of purified c-Src at its primary negative regulation site. J. Biol. Chem. 270:25729–25732.[Abstract/Free Full Text]
  38. Frame M.C. (2002) Src in cancer: deregulation and consequences for cell behaviour. Biochim. Biophys. Acta 1602:114–130.[Medline]
  39. Gartel A.L. and Tyner A.L. (2002) The role of the cyclin-dependent kinase inhibitor p21 in apoptosis. Mol. Cancer Ther. 1:639–649.[Abstract/Free Full Text]
  40. Ashok B.T., Chen Y.G., Liu X., Garikapaty V.P., Seplowitz R., Tschorn J., Roy K., Mittelman A., Tiwari R.K. (2002) Multiple molecular targets of indole-3-carbinol, a chemopreventive anti-estrogen in breast cancer. Eur. J. Cancer Prev. 11:S86–S93.
  41. Cunliffe H.E., Ringner M., Bilke S., Walker R.L., Cheung J.M., Chen Y., Meltzer P.S. (2003) The gene expression response of breast cancer to growth regulators: patterns and correlation with tumor expression profiles. Cancer Res. 63:7158–7166.[Abstract/Free Full Text]
  42. Bianco R., Shin I., Ritter C.A., Yakes F.M., Basso A., Rosen N., Tsurutani J., Dennis P.A., Mills G.B., Arteaga C.L. (2003) Loss of PTEN/MMAC1/TEP in EGF receptor-expressing tumor cells counteracts the antitumor action of EGFR tyrosine kinase inhibitors. Oncogene 22:2812–2822.[CrossRef][ISI][Medline]
  43. Okubo S., Kurebayashi J., Otsuki T., Yamamoto Y., Tanaka K., Sonoo H. (2004) Additive antitumour effect of the epidermal growth factor receptor tyrosine kinase inhibitor gefitinib (Iressa, ZD1839) and the antioestrogen fulvestrant (Faslodex, ICI 182,780) in breast cancer cells. Br. J. Cancer 90:236–244.[CrossRef][ISI][Medline]
  44. Bao J., Gur G., Yarden Y. (2003) Src promotes destruction of c-Cbl: implications for oncogenic synergy between Src and growth factor receptors. Proc. Natl Acad. Sci. USA 100:2438–2443.[Abstract/Free Full Text]
  45. Manson M.M., Hudson E.A., Ball H.W., Barrett M.C., Clark H.L., Judah D.J., Verschoyle R.D., Neal G.E. (1998) Chemoprevention of aflatoxin B1-induced carcinogenesis by indole-3-carbinol in rat liver - predicting the outcome using early biomarkers. Carcinogenesis 19:1829–1836.[Abstract/Free Full Text]
  46. Chatterji U., Riby J.E., Taniguchi T., Bjeldanes E.L., Bjeldanes L.F., Firestone G.L. (2004) Indole-3-carbinol stimulates transcription of the interferon gamma receptor 1 gene and augments interferon responsiveness in human breast cancer cells. Carcinogenesis 25:1119–1128.[Abstract/Free Full Text]
  47. Ishizawar R. and Parsons S.J. (2004) c-Src and cooperating partners in human cancer. Cancer Cell 6:209–214.[CrossRef][ISI][Medline]
  48. Cabodi S., Moro L., Baj G., et al. (2004) p130Cas interacts with estrogen receptor alpha and modulates non-genomic estrogen signaling in breast cancer cells. J. Cell. Sci. 117:1603–1611.[Abstract/Free Full Text]
  49. Laskin J.J. and Sandler A.B. (2004) Epidermal growth factor receptor: a promising target in solid tumours. Cancer Treat Rev. 30:1–17.[CrossRef][ISI][Medline]
  50. Bild A.H., Yao G., Chang J.T., et al. (2006) Oncogenic pathway signatures in human cancers as a guide to targeted therapies. Nature 439:353–357.[CrossRef][Medline]
  51. Furstoss O., Manes G., Roche S. (2002) Cyclin E and cyclin A are likely targets of Src for PDGF-induced DNA synthesis in fibroblasts. FEBS Lett. 526:82–86.[CrossRef][ISI][Medline]
  52. Lo H.W. and Hung M.C. (2006) Nuclear EGFR signalling network in cancers: linking EGFR pathway to cell cycle progression, nitric oxide pathway and patient survival. Br. J. Cancer 94:184–188.[CrossRef][ISI][Medline]
  53. Armstrong D.K., Kaufmann S.H., Ottaviano Y.L., Furuya Y., Buckley J.A., Isaacs J.T., Davidson N.E. (1994) Epidermal growth factor-mediated apoptosis of MDA-MB-468 human breast cancer cells. Cancer Res. 54:5280–5283.[Abstract/Free Full Text]
  54. Fan Z., Lu Y., Wu X., DeBlasio A., Koff A., Mendelsohn J. (1995) Prolonged induction of p21Cip1/WAF1/CDK2/PCNA complex by epidermal growth factor receptor activation mediates ligand-induced A431 cell growth inhibition. J. Cell. Biol. 131:235–242.[Abstract/Free Full Text]
  55. Chin Y.E., Kitagawa M., Kuida K., Flavell R.A., Fu X.Y. (1997) Activation of the STAT signaling pathway can cause expression of caspase 1 and apoptosis. Mol. Cell. Biol. 17:5328–5337.[Abstract]
  56. Kumar R.R., Meenakshi A., Sivakumar N. (2001) Enzyme immunoassay of human epidermal growth factor receptor (hEGFR). Hum. Antibodies 10:143–147.[Medline]
  57. Tidow N., Boecker A., Schmidt H., Agelopoulos K., Boecker W., Buerger H., Brandt B. (2003) Distinct amplification of an untranslated regulatory sequence in the egfr gene contributes to early steps in breast cancer development. Cancer Res. 63:1172–1178.[Abstract/Free Full Text]
Received April 28, 2006; revised July 31, 2006; accepted September 1, 2006.


Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 Molecular Cancer TherapeuticsHome page
E. P. Moiseeva, G. M. Almeida, G. D.D. Jones, and M. M. Manson
Extended treatment with physiologic concentrations of dietary phytochemicals results in altered gene expression, reduced growth, and apoptosis of cancer cells
Mol. Cancer Ther., November 1, 2007; 6(11): 3071 - 3079.
[Abstract] [Full Text] [PDF]

<
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
28/2/435    most recent
bgl171v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal