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Endocrine-Related Cancer 12 (Supplement_1) S75 -84     DOI: 10.1677/erc.1.01023
Copyright © 2005 by the Society for Endocrinology
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Elevated ERK1/ERK2/estrogen receptor cross-talk enhances estrogen-mediated signaling during long-term estrogen deprivation

Lesley-Ann Martin1,4, Ian Farmer1,4, Stephen R D Johnston2, Simak Ali3 and Mitch Dowsett1,4

1 Molecular Endocrinology, Breakthrough Breast Cancer Centre, Institute of Cancer Research, Chester Beatty Laboratories, Fulham Rd, London SW3 6JB, UK
2 Breast Unit, Royal Marsden Hospital, Fulham Rd, London SW3 6JJ, UK
3 Department of Medicine, Imperial College School of Medicine, Hammersmith Hospital, London, UK
4 Academic Department of Biochemistry, Institute of Cancer Research, Royal Marsden Hospital, Fulham Rd, London, SW3 6JJ, UK

(Requests for offprints should be addressed to Lesley-Anne Martin; Email: lesley-ann.martin{at}icr.ac.uk)

This paper was presented at the 1st Tenovus/AstraZeneca Workshop, Cardiff (2005). AstraZeneca has supported the publication of these proceedings.


   Abstract
Top
 Abstract
Introduction
 LTED cell lines are...
 Role of ERK1/ERK2 in...
 Increased ERBB2 is associated...
 pERK1/ERK2 does not play...
 Targets for therapeutic...
 References
 
The knowledge that steroids play a pivotal role in the development of breast cancer has been exploited clinically by the development of endocrine treatments. These have sought to perturb the steroid hormone environment of the tumour cells, predominately by withdrawal or antagonism of oestrogen. Unfortunately, the beneficial actions of existing endocrine treatments are attenuated by the ability of tumours to circumvent the need for steroid hormones, whilst in most cases, retaining the nuclear steroid receptors. The mechanisms involved in resistance to estrogen deprivation are of major clinical relevance for optimal treatment of breast cancer patients and the development of new therapeutic regimes. We have shown that long-term culture of MCF7 cells in medium depleted of oestrogen (LTED) results in hypersensitivity to oestradiol (E2) coinciding with elevated levels of both ER{alpha} phosphorylated on Ser118 and ERK1/ERK2. Our data suggest elevated ERK1/ERK2 activity results wholly or in part from enhanced ERBB2 expression in the LTED cells. These cells showed greater sensitivity to the tyrosine kinase inhibitor ZD1839 in both ER{alpha}-mediated transcription and growth assays compared with the wt-MCF7. Similarly the MEK inhibitor U0126 decreased basal ER{alpha}-mediated transcription and proliferation in the LTED cells by 50% and reduced their sensitivity to the proliferative effects of E2 10-fold, whilst having no effect on the wild type (wt). However, complete suppression of ERK1/ERK2 activity in the LTED cells did not inhibit ER{alpha} Ser118 phosphorylation suggesting that the cells remained ligand-dependent. This was further confirmed by the increased sensitivity of the LTED cells to the growth suppressive effects of ICI 182,780 and suggested that the LTED cells remained wholly or partially dependent on oestrogen receptor (ER)/oestrogen responsive elements directed growth. These findings suggest that treatments targeted at growth factor signalling pathways may be useful in patients acquiring resistance to oestrogen deprivation with aromatase inhibitors and that the pure anti-oestrogen ICI 182,780 may also be effective by blocking or destabilizing ER and hence disrupting cross-talk.


   Introduction
Top
 Abstract
 Introduction
LTED cell lines are...
 Role of ERK1/ERK2 in...
 Increased ERBB2 is associated...
 pERK1/ERK2 does not play...
 Targets for therapeutic...
 References
 
Breast cancer is the most common cancer in women in the western world, with approximately 40 000 new cases diagnosed each year in the UK alone. Oestrogens play a pivotal role in the development of breast cancer and exert their effects by binding to the oestrogen receptor (ER). Oestradiol (E2)-bound ER interacts with oestrogen response elements (ERE) regulating transcription on target genes controlling both proliferation and cell survival. This knowledge has led to the development of endocrine therapies that reduce E2 activity either by blocking its biosynthesis using aromatase inhibitors or competing with E2 for the ER with agents such as the selective ER modulator tamoxifen. Despite the success of current endocrine agents the majority of women eventually relapse whilst maintaining functional steroid receptors. Cross-talk between the receptor tyrosine kinase (RTK) and ER signal transduction pathways may contribute to acquired endocrine resistance by sensitising breast tumor cells to oestrogens or by circumventing the need for hormone. Based on this evidence one strategy to improve the efficacy of current endocrine agents as well as delaying the onset of resistance is to target the ER and RTK signaling pathway concomitantly.

In an attempt to elucidate these mechanisms, our laboratory and others have developed in vitro models to study the molecular changes associated with longterm oestrogen deprivation (LTED) (Masamura et al. 1995, Coutts & Murphy 1998, Chan et al. 2002). In the following manuscript we review the current findings and compare these with our own and propose the various treatment regimes that may be on offer for patients who have relapsed with acquired resistance to LTED.


   LTED cell lines are hypersensitive to E2 and remain sensitive to the pure anti-oestrogen ICI 182,780
Top
 Abstract
 Introduction
 LTED cell lines are...
Role of ERK1/ERK2 in...
 Increased ERBB2 is associated...
 pERK1/ERK2 does not play...
 Targets for therapeutic...
 References
 
To develop a model representative of relapse after LTED we cultured the human breast tumour cell line MCF-7 in E2 depleted medium for 80 weeks taking weekly samples for phenotypic analysis. The cell line passed through an initial quiescent phase lasting approximately 12 weeks followed by an increase in basal cell proliferation. At this stage the LTED cells were shown to be hypersensitive to the addition of exogenous E2 with doses as low as 10–13M (Fig. 1AGo) causing a marked increase in proliferation compared with the parental (wild type, wt) MCF-7 (Masamura et al. 1995, Jeng et al. 1998, Chan et al. 2002, Yue et al. 2002, Martin et al. 2003). It should be noted that while it is reasonable to describe this response to low doses of E2 as hypersensitivity it does not comply with a strict definition of a significantly reduced dose required to achieve 50% maximal stimulation.



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  		Figure 1 Long-term oestrogen deprivation (LTED) results in hypersensitivity to exogenous estradiol (E2) coupled with enhanced oestrogen receptor (ER)/oestrogen response elements (ERE) directed basal transcription. (A) LTED and wild type (wt) MCF-7 cells (depleted of E2 for 5 days ) were treated with increasing doses of E2 for 6 days. (B) Basal ER mediated transcription is elevated in the LTED cells compared with the wt MCF-7. Cells were co-transfected with EREIItkLuc and pCH110. After transfection cells were treated with DCC-FBS medium for 24 h. Cells were harvested and luciferase and ß-galactosidase activities measured. To correct for differences in transfection efficiency, the luciferase activities were normalised to ß-galactosidase activities. (C) The effect of E2 on ER-mediated transcription. Wt and LTED were treated as described above followed by 24 h incubation with DCC-FBS medium containing increasing doses of E2. Normalized luciferase activity was expressed relative to the vehicle treated control. Error bars represent means ± S.E.M.

 
Of particular note, doses in excess of 10–10M were inhibitory for the LTED cells whilst providing maximum stimulation for the wild type (wt) MCF-7. Studies have recently shown that in the presence of high doses of E2 there is a concomitant increase in the expression of Fas ligand. As a consequence it has been postulated that tumour regression in response to high doses of E2 may result from Fas mediated apoptosis (Song et al. 2001, Osipo et al. 2003). Under such circumstances, treating patients who have relapsed on aromatase inhibitors with high doses of E2 may exploit this phenomenon providing clinical benefit.

Further analysis of our LTED cell line showed a marked elevation in ER{alpha} which was phosphorylated on serine 118 (Ser118) in the absence of E2. Assessment of the basal ER/ERE activity showed a 10-fold elevation compared with the wt MCF-7 (Fig. 1BGo). Treatment of both cell lines with increasing doses of E2 revealed a dose-dependent elevation in ER-mediated transcription. However, as the LTED cells have a higher level of basal transcription we believe that they show a marked degree of hypersensitivity to E2. This is particularly evident at 10–13M E2 where the LTED cells transcription is approximately 10-fold higher than the wt MCF-7 cells in absolute terms (Fig. 1CGo).

Treatment of the LTED cell line with escalating doses of the pure anti-oestrogen ICI 182,780 showed a marked dose dependent sensitivity similar in profile to the wt MCF-7 cells in the presence of E2 (Fig. 2AGo). This was reciprocated in ER/ERE reporter assays where LTED cells showed a marked sensitivity to ICI 182,780 whilst the wt cell line remained unaffected in the absence of ligand (Fig. 2BGo). As studies have suggested that ICI 182,780 may impact directly on growth factor pathways independent of ER (Huynh et al. 1996, Salerno et al. 1999, Chan et al. 2001) we postulated that if this were the case addition of E2 would be unable to overcome the inhibitory effects of ICI 182,780. However, treatment of the LTED cells with a standard inhibitory dose of ICI 182,780 and increasing doses of E2 was able to rescue the cells, negating the possibility that ICI 182,780 was directly inhibiting growth factor pathways (Fig. 2CGo). Taken together these data suggested that the LTED cells use a classical ER/ERE directed process either wholly or partially in their adaptive process.



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  		Figure 2 The pure anti-oestrogen ICI 182,780 inhibits LTED cell growth. (A) LTED and wt MCF-7 cells were cultured in the presence of increasing doses of ICI 182,780 alone in the case of LTED cells or in combination with 10–9M E2 for wt MCF7. Cells were incubated for 6 days. Cell number was established using a coulter counter. Data is expressed compared with control. (B) ICI 182,780 inhibits basal ER-mediated transcription in LTED cells compared with wt MCF-7. Wt and LTED cells were transiently co-transfected with EREIItkLuc and pCH110 in serum free medium followed by 24 h incubation with DCC medium containing increasing doses of ICI 182,780. Normalized luciferase activity from triplicate wells was expressed relative to the vehicle treated control. (C) LTED cells were cultured as previously described in the presence of 10–8M ICI 182,780 and increasing doses of E2. Error bars represent means ± S.E.M.

 

   Role of ERK1/ERK2 in the LTED phenotype
Top
 Abstract
 Introduction
 LTED cell lines are...
 Role of ERK1/ERK2 in...
Increased ERBB2 is associated...
 pERK1/ERK2 does not play...
 Targets for therapeutic...
 References
 
Several studies have proposed a role for the ERK signalling pathway in the initiation and pathogenesis of breast cancer (Sivaraman et al. 1997). Activation of the ERK cascades modulate the phosphorylation and hence activity of several nuclear transcription factors which in turn regulate genes involved in proliferation and cell survival.

ER{alpha}, is functionally regulated via phosphorylation by several protein kinases (reviewed by Ali & Coombes 2002). Phosphorylation of Ser118 is mediated by cdk7 in response to E2 and is also phosphorylated by pERK1/ERK2 in a ligand-independent manner (Bunone et al. 1996, Chen et al. 2000). Serine 167 (Ser167) on the other hand is the target for AKT and p90RSK, which is activated by pERK1/ERK2. Hence increased ERK1/ERK2 activity could result in endocrine resistance. Analysis of our LTED cell line showed an increase in both activated ERK1/ERK2 (Coutts & Murphy 1998, Jeng et al. 2000) and pp90RSK (Martin et al. 2003) coinciding with the onset of hypersensitivity (Fig. 3AGo). We postulated that ERK1/ERK2 was playing an integral role in the adaptation of the LTED cells. To test this we cultured the wt MCF-7 and LTED cells in the presence of escalating doses of the MEK 1/2 inhibitor UO126. Whilst the wt cells were largely unaffected the proliferation and ER/ERE directed transcription within the LTED cells was reduced (Figs 3B & CGo).



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  		Figure 3 Increased ERK1/2 activity is associated with the LTED phenotype. (A) Whole cell extracts (50 µg) from LTED and wt MCF-7 cultured under basal conditions were separated through 10% SDS-PAGE gels and immunoprobed with antibodies specific for the proteins shown above. (B) The Effect of MEK inhibitor U0126 on the Growth of LTED and wt MCF-7 cells. LTED and wt cells were seeded into 12-well plates at a density of 1 x 104 per well. After 48 h the cells were treated with 5 or 10 µM U0126 over a period of 6 days. Cell number was determined using a coulter counter. Western blots show the level of phosphorylated pERK1/ERK2 inhibition with increasing doses of U0126. (C) The effect of MEK inhibitor U0126 on basal ER{alpha} transactivation. Wt and LTED cells were transiently co-transfected with EREIItkLuc and pCH110 in serum free medium followed by 24 h incubation in DCC-FBS medium containing U0126. Normalized luciferase activity was expressed relative to the vehicle treated control. (D) The effect of MEK inhibitor U0126 on oestradiol sensitivity. Cells were seeded at 1 x 104 per well. After 48 hours cells were treated with DCC-FBS medium ± U0126 and increasing doses of E2. Error bars represent means ± S.E.M.

 
At this stage we postulated that pERK1/ERK2 was sensitising the cells to the residual E2 in the stripped medium. To test this hypothesis LTED cells were cultured in the presence of increasing doses of E2 ± an inhibitory dose of UO126. In the presence of UO126 the dose response to E2 shifted to the right by almost two logs providing a proliferation profile similar to the wt MCF-7 response to exogenous E2 (Fig. 3DGo). Despite pERK1/ERK2 being completely blocked by the inhibitor, both transcription and proliferation were only reduced by 50% suggesting that alternate pathways were also in operation. As the PI3 kinase pathway has also been implicated in resistance (reviewed by Ali & Coombes 2002) we assessed the LTED cells for the expression of AKT. Although no marked elevation was noted, treatment of the LTED cells with the PI3 kinase inhibitor LY294002 resulted in a 70% decrease in basal transcription whilst having no effect on the wt MCF-7 in the absence of exogenous E2. However, in the presence of E2, LY294002 was inhibitory to both cell lines and although increasing doses of E2 were able to rescue the inhibitory effects of LY294002 in the LTED cell line, the wt remained inhibited. Treatment of the cells with a combination of the inhibitors had a marked inhibitory effect on both the wt (50%) and LTED (70%), and whilst addition of E2 (10–9M) was unable to remove the inhibitory effects in the wt it was able to partially restore ER{alpha} transactivation in the LTED (Martin et al. 2003). The findings suggest that the enhanced activity of the MAP kinase pathway only partially explains the hypersensitivity to E2 and that the AKT pathway is not significantly involved; other pathways such as the mToR pathway are being investigated to identify the additional source of resistance.


   Increased ERBB2 is associated with the LTED phenotype
Top
 Abstract
 Introduction
 LTED cell lines are...
 Role of ERK1/ERK2 in...
 Increased ERBB2 is associated...
pERK1/ERK2 does not play...
 Targets for therapeutic...
 References
 
The question that remained unanswered was the mode of action leading to the elevation in pERK1/ERK2. Although ER is classically genomic in its action, several studies have implicated ER in non-genomic effects and demonstrated the ability of oestrogens mediated by ER to activate the ERK1/ERK2 pathway (Miglaccio et al. 1996, Castoria et al. 1999, Kousteni et al. 2002, Song et al. 2002). Santen’s group have concluded that activated pERK1/ERK2 in their LTED setting results via a non-genomic mechanism whereby ER binds to the shc adaptor protein which is then anchored at the membrane by its interaction with IGF-1R (Song et al. 2002, Song et al. 2004). To ascertain if we could also see rapid activation of ERK1/ERK2 in our LTED setting we treated the LTED and wt MCF-7 cells with E2 over a 2 hour time course and although we saw rapid phosphorylation of ER{alpha} Ser118, this was unassociated with an increase in pERK1/pERK2 activity (Joel et al. 1998, Lobenhofer & Marks 2000, Lobenhofer et al. 2000, Martin et al. 2003). This apparent discrepancy is likely to be a result of variations in cell lines and also experimental conditions.

Further analysis of our own LTED cells revealed an elevation in ERBB2 activity (Fig. 4AGo). Previous studies have shown that elevated levels of epidermal growth factor receptor (EGFR) and ERBB2 are associated with tamoxifen resistant MCF-7 cells and that these cells are more sensitive to the anti-proliferative effects of ZD1839 compared with wt MCF-7 cells (Nicholson et al. 2001, Knowlden et al. 2003). Treatment of the LTED cells with ZD1839 also revealed enhanced sensitivity compared with the wt MCF-7 both in proliferation and ERE reporter assays. LTED cells had an IC50 of approximately 5 µM in keeping with an ERBB2-dependent rather than EGFR-dependent effect (Fig. 4B & CGo). Fluorescence in-situ hybridization (FISH) analysis indicated that ERBB2 was not amplified in the LTED cells suggesting the up-regulation maybe via a transcriptional mechanism. Taken together these data suggest that elevations in ERBB2 may be wholly or partially responsible for increased pERK1/ERK2 activity in this setting. Further work using molecular or pharmacological antagonists of ERBB2 are needed to reveal the degree of involvement. It is notable that IGF-1R levels are 2–3-fold enhanced over wt levels in the LTED cells (L-A Martin, S Pancholi, SRD Johnston & M Dowsett, unpublished observations) and this may interact with the increased signalling seen from ERBB2.



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  		Figure 4 ERBB2 is elevated during LTED. (A) The level of phosphorylated and total ERBB2 in the LTED cells. Whole cell extracts from wt MCF-7 versus the LTED cell line were immunoprobed with antibodies specific for phosphorylated (Y1248) and total ERBB2. (B) The effect of ZD1839 on LTED, wt MCF7 and SKBR3 cell growth. Wt MCF-7, LTED and SKBR3 cells were seeded at a density of 5 x 103 cells per well and 48 h later treated with increasing doses of ZD1839, a specific inhibitor of epidermal growth factor receptor (EGFR). Wt cells were cultured in the presence of 10–9M E2 whilst LTED cells were grown in DCC-FBS medium. SKBR3 cells were cultured in RPMI 1640 containing phenol red and 10% FBS. (C) The effect of EGFR inhibitor ZD1839 on basal and E2-mediated ER{alpha} transactivation. Wt and LTED cells were transiently co-transfected with EREIItkLuc and pCH110 in serum free medium followed by 24 h incubation in DCC ± ZD1839. Normalised luciferase activity was expressed relative to the vehicle treated control. Error bars represent means ± S.E.M.

 

   pERK1/ERK2 does not play a role in the phosphorylation of ER{alpha} Ser118
Top
 Abstract
 Introduction
 LTED cell lines are...
 Role of ERK1/ERK2 in...
 Increased ERBB2 is associated...
 pERK1/ERK2 does not play...
Targets for therapeutic...
 References
 
As described previously pERK1/2 has been associated with the ligand-independent phosphorylation of the ER{alpha} Ser118. Although our study suggested that the LTED cells remained ligand-dependent it was impossible to ignore the fact that both pERK1/2 and ER{alpha} Ser118 were elevated in the LTED phenotype. To eliminate pERK1/ERK2 in the activation of ER{alpha} Ser118 we treated both wt and LTED cells with the MEK inhibitor UO126 ± E2 (Fig. 5Go) or LY294002 ± E2 as the ERK pathway can be activated in a MEK1/2 independent manner via PI3 kinase and protein kinase C (data not shown) (Grammer & Blenis 1997). In both cases neither kinase was responsible for phosphorylation of ER{alpha} Ser118 adding further evidence to the ligand dependent nature of the LTED cell line. Taken together these data suggested that a complex interplay between the two signaling pathways was responsible for the LTED phenotype. We postulate that although ERK1/ERK2 is not responsible for direct phosphorylation of the ER, that it plays a pivotal role by activating the transcription machinery leading to phosphorylation of co-activators of the p160 family such as amplified in breast 1 (AIB1) (Font de Mora & Brown 2000) whilst downstream partners such as p90RSK can phosphorylate and enhance the activity of CREB binding protein (CBP) (Nakajima et al. 1996) together with other factors associated with the basal transcription machinery. These findings may account for the elevated basal transcription in the LTED cell line.



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  		Figure 5 The Effect of U0126 on ER{alpha} Ser118 phosphorylation. LTED and wt cells were serum starved for 24 h prior to experimentation. Cell monolayers were pre-treated with U0126 (20 µM) or vehicle for 30 min, followed by treatment with 10–8M E2, U0126 or a combination of the two for a further 30 min. Cells were harvested and the level of pERK1/ERK2 activity and Ser118 phosphorylation was monitored by immunoblotting.

 

   Targets for therapeutic intervention
Top
 Abstract
 Introduction
 LTED cell lines are...
 Role of ERK1/ERK2 in...
 Increased ERBB2 is associated...
 pERK1/ERK2 does not play...
 Targets for therapeutic...
References
 
In summary we have shown that the LTED cells remain ligand dependent and are hypersensitive to E2 requiring ER for both proliferation and transcription. These data suggest that ICI 182,780 might be more effective in the treatment of breast cancers that acquire resistance to oestrogen deprivation. Based upon these results we recently initiated a clinical trial for the treatment of patients with advanced breast cancer. Patients are randomised to receive ICI 182,780 (fulvestrant), exemstane or anastrozole in combination with ICI 182,780 (the SoFEA trial).

Although we have shown that elevation in ERK1/2 activity (possibly via elevated ERBB2 expression) plays an integral role in the development of the LTED phenotype and sensitisation of these cells to residual E2, it is not responsible for the phosphorylation of ER{alpha} Ser118. We postulate an adaptive pathway similar to that shown in Fig. 6Go in which the development of the LTED phenotype results from elevated levels of ER coupled with enhanced activation of the ER as a result of increased ERBB2 expression and pERK1/ERK2 activity. pERK1/ERK2 may be involved in ER activation (on sites other than Ser118) and regulation of down-stream partners such as p90RSK and AIB1 could lead to increased coactivator activity providing a hypersensitive reception to residual E2.



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  		Figure 6 Possible cross-talk between the ERBB2 receptor and oestrogen receptor during LTED. We postulate multiple phosphorylation events involving the PI3 kinase and MAPK pathways sensitise the LTED cells to low levels of E2, by activating the ER and possible coactivators (bold lines). Based on current literature (reviewed by Ali & Coombes 2002) the pathways involved in acquired endocrine resistance are shown together with potential targets for drug intervention.

 
In conclusion these data confirm the presence of cross-talk between the ER and growth factor signalling pathways during LTED. Based on this evidence one strategy to improve the efficacy of current endocrine agents as well as delaying the onset of resistance is to target the ER and RTK signaling pathway concomitantly (Fig. 6Go). Studies have shown that treatment with ZD1839 can re-sensitise tamoxifen resistant MCF-7 cells to the anti-proliferative effects of tamoxifen prolonging the usefulness of the drug (Nicholson et al. 2002). In a similar study combined use of tamoxifen and ZD1839 delayed development of tamoxifen resistance (Gee et al. 2003). We recently carried out a similar study to determine whether AEE788 (a combined inhibitor of the EGFR/human epidermal growth factor receptor 2 (HER2) and vascular endothelial growth factor (VEGF) tyrosine kinases) together with tamoxifen or letrozole would enhance the anti-proliferative effects of these agents. Human breast cancer cell lines with varying expression of ER and HER2 [MCF7 (ER+HER2–), ZR75.1 (ER+HER2+), BT474 (ER+, HER2++) and SKBR3 (ER– , HER2++)] were engineered to express aromatase. Clones were screened in proliferation assays for response to E2, androstenedione (AND), 4OH-tamoxifen (4OH-T), letrozole (LET) and AEE788, alone or in combination. Both E2 and AND induced a proliferative response in ER+ cell lines in a dose-dependent manner. Similarly, ER+ cell lines were inhibited by 4OH-T or LET (IC50 for 4OH-T: approximately 10nm for MCF7, ZR75.1 and BT474; IC50 for LET: 5, 1 and 5 nM respectively. The effect of AEE788 on proliferation varied between cell lines according to their EGFR/HER2 status (IC50 of approx. 5–10, 2–5, 1.5 and 0.5 µM for MCF7, ZR75.1, SKBR3 and BT474 respectively). Combinations of AEE788 with 4OH-T or LET enhanced the anti-proliferative effects of these agents (MCF7 and ZR75.1, 20–30% and BT474, 60–70%) via an additive selective mechanism (Hauge Evans et al. 2004). These data suggest that combinations of AEE788 with tamoxifen or letrozole in breast cancer over expressing EGFR and/or HER2 may provide superior anti-tumour activity as compared with the single agents. There is now enhanced interest in the application of endocrine agents in combination with signal transduction inhibitors which selectively block growth factor receptors and their downstream partners, such as ERK, AKT, mTOR and farnesyltransferase activity (Fig. 6Go) with several clinical trials currently underway (reviewed by Johnston et al. this issue).


   References
Top
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 Introduction
 LTED cell lines are...
 Role of ERK1/ERK2 in...
 Increased ERBB2 is associated...
 pERK1/ERK2 does not play...
 Targets for therapeutic...
 References
 
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