Correction to: Heregulin ß1 drives gefitinib-resistant growth and invasion in tamoxifen-resistant MCF-7 breast cancer cells.

After the publication of this work [1], an error was noticed in Fig. 2b and Fig. 4b as well as Fig. 4b. and Fig. 5d. Images of the ERK1/2 blots were accidentally duplicated. In Fig. 5a. and Fig. 5c., the last lane for p-ERK1/2 was mistakenly cropped out of the final image. The original blot for Fig. 4b., "total EGFR" (or lane 2) is shown below to avoid any misunderstanding of the data. We apologize for this error, which did not affect any of the interpretations or conclusions of the article.

Impact of dual mTORC1/2 mTOR kinase inhibitor AZD8055 on acquired endocrine resistance in breast cancer in vitro.

INTRODUCTION: Upregulation of PI3K/Akt/mTOR signalling in endocrine-resistant breast cancer (BC) has identified mTOR as an attractive target alongside anti-hormones to control resistance. RAD001 (everolimus/Afinitor®), an allosteric mTOR inhibitor, is proving valuable in this setting; however, some patients are inherently refractory or relapse during treatment requiring alternative strategies. Here we evaluate the potential for novel dual mTORC1/2 mTOR kinase inhibitors, exemplified by AZD8055, by comparison with RAD001 in ER + endocrine resistant BC cells. METHODS: In vitro models of tamoxifen (TamR) or oestrogen deprivation resistance (MCF7-X) were treated with RAD001 or AZD8055 alone or combined with anti-hormone fulvestrant. Endpoints included growth, cell proliferation (Ki67), viability and migration, with PI3K/AKT/mTOR signalling impact monitored by Western blotting. Potential ER cross-talk was investigated by immunocytochemistry and RT-PCR. RESULTS: RAD001 was a poor growth inhibitor of MCF7-derived TamR and MCF7-X cells (IC50 ≥1 µM), rapidly inhibiting mTORC1 but not mTORC2/AKT signalling. In contrast AZD8055, which rapidly inhibited both mTORC1 and mTORC2/AKT activity, was a highly effective (P <0.001) growth inhibitor of TamR (IC50 18 nM) and MCF7-X (IC50 24 nM), and of a further T47D-derived tamoxifen resistant model T47D-tamR (IC50 19 nM). AZD8055 significantly (P <0.05) inhibited resistant cell proliferation, increased cell death and reduced migration. Furthermore, dual treatment of TamR or MCF7-X cells with AZD8055 plus fulvestrant provided superior control of resistant growth versus either agent alone (P <0.05). Co-treating with AZD8055 alongside tamoxifen (P <0.01) or oestrogen deprivation (P <0.05) also effectively inhibited endocrine responsive MCF-7 cells. Although AZD8055 inhibited oestrogen receptor (ER) ser167 phosphorylation in TamR and MCF7-X, it had no effect on ER ser118 activity or expression of several ER-regulated genes, suggesting the mTOR kinase inhibitor impact was largely ER-independent. The capacity of AZD8055 for ER-independent activity was further evidenced by growth inhibition (IC5018 and 20 nM) of two acquired fulvestrant resistant models lacking ER. CONCLUSIONS: This is the first report demonstrating dual mTORC1/2 mTOR kinase inhibitors have potential to control acquired endocrine resistant BC, even under conditions where everolimus fails. Such inhibitors may prove of particular benefit when used alongside anti-hormonal treatment as second-line therapy in endocrine resistant disease, and also potentially alongside anti-hormones during the earlier endocrine responsive phase to hinder development of resistance.

erbB3 recruitment of insulin receptor substrate 1 modulates insulin-like growth factor receptor signalling in oestrogen receptor-positive breast cancer cell lines.

INTRODUCTION: Recently we reported that insulin receptor substrate 1 (IRS-1), classically an adaptor protein for the insulin-like growth factor type I receptor (IGF-IR), associates with the epidermal growth factor receptor in oestrogen receptor (ER)-positive (ER+) tamoxifen-resistant breast cancer cells. In this study, we examined whether IRS-1 also associates with another erbB receptor family member, erbB3, and what impact this might have on IGF-IR signalling in three ER+ breast cancer cell lines. METHODS: Immunoprecipitation and Western blot analysis were utilised to examine the potential association between erbB3 and IRS-1 in MCF-7, T47D and BT-474 cells in the absence and presence of the erbB3/4 ligand heregulin ß1 (HRGß1). Subsequently, the impact of a selective IGF-IR/IR inhibitor 4-anilino-5-bromo-2-[4-(2-hydroxy-3-(N, N-dimethylamino)propoxy)anilino]pyrimidine on this association and HRGß1 signalling was assessed in these cell lines. Immunohistochemical analysis of a small cohort of ER+ breast cancer patient samples was also performed to determine the potential clinical relevance of this novel interaction. RESULTS: Immunoprecipitation and Western blot analysis revealed an interaction between erbB3 and IRS-1 in MCF-7, T47D and BT-474 cells, with HRGß1 significantly enhancing this recruitment and promoting IRS-1 phosphorylation at Y612. IRS-1 participates in erbB3 signalling in MCF-7 and T47D cells as IRS-1 knockdown impaired HRGß1 signalling. Importantly, recruitment of IRS-1 by erbB3 reduced IRS-1 association with IGF-IR in MCF-7 and T47D cells, whilst blockade of IGF-IR-enhanced erbB3-IRS-1 interaction and sensitised both cell lines to HRGß1, allowing HRGß1 to override IGF-IR blockade. Consequently, suppression of IRS-1 signalling enhanced the effects of IGF-IR inhibition in these cells. This novel interaction may have clinical relevance, as immunohistochemical analysis of a small ER+ breast tumour series revealed significant positive correlations between phosphorylated IRS-1 Y612 expression and total erbB3, phosphorylated Akt and Ki-67 expression. CONCLUSIONS: IRS-1 can be recruited to IGF-IR and erbB3 in ER+ breast cancer cells, and this provides an adaptive resistance mechanism when these receptors are targeted individually. Consequently, cotargeting IGF-IR and either erbB3 or IRS-1 should prove to be a more effective strategy for the treatment of ER+ breast cancer.

Fulvestrant-induced expression of ErbB3 and ErbB4 receptors sensitizes oestrogen receptor-positive breast cancer cells to heregulin ß1.

INTRODUCTION: We have previously reported that induction of epidermal growth factor receptor and ErbB2 in response to antihormonal agents may provide an early mechanism to allow breast cancer cells to evade the growth-inhibitory action of such therapies and ultimately drive resistant cell growth. More recently, the other two members of the ErbB receptor family, ErbB3 and ErbB4, have been implicated in antihormone resistance in breast cancer. In the present study, we have investigated whether induction of ErbB3 and/or ErbB4 may provide an alternative resistance mechanism to antihormonal action in a panel of four oestrogen receptor (ER)-positive breast cancer cell lines. METHODS: MCF-7, T47D, BT474 and MDAMB361 cell lines were exposed to fulvestrant (100 nM) for seven days, and effects on ErbB3/4 expression and signalling, as well as on cell growth, were assessed. Effects of heregulin ß1 (HRGß1) were also examined in the absence and presence of fulvestrant to determine the impact of ER blockade on the capacity of this ErbB3/4 ligand to promote signalling and cell proliferation. RESULTS: Fulvestrant potently reduced ER expression and transcriptional activity and significantly inhibited growth in MCF-7, T47D, BT474 and MDAMB361 cells. However, alongside this inhibitory activity, fulvestrant also consistently induced protein expression and activity of ErbB3 in MCF-7 and T47D cells and ErbB4 in BT474 and MDAMB361 cell lines. Consequently, fulvestrant treatment sensitised all cell lines to the actions of the ErbB3/4 ligand HRGß1 with enhanced ErbB3/4-driven signalling activity, reexpression of cyclin D1 and significant increases in cell proliferation being observed when compared to untreated cells. Indeed, in T47D and MDAMB361 HRGß1 was converted from a ligand having negligible or suppressive growth activity into one that potently promoted cell proliferation. Consequently, fulvestrant-mediated growth inhibition was completely overridden by HRGß1 in all four cell lines. CONCLUSIONS: These findings suggest that although antihormones such as fulvestrant may have potent acute growth-inhibitory activity in ER-positive breast cancer cells, their ability to induce and sensitise cells to growth factors may serve to reduce and ultimately limit their inhibitory activity.

Antihormone induced compensatory signalling in breast cancer: an adverse event in the development of endocrine resistance.

Using MCF7 breast cancer cells, it has been shown that antihormones promote expression/activity of oestrogen-repressed tyrosine kinases, notably EGFR, HER2 and Src. These inductive events confer responsiveness to targeted inhibitors (e.g., gefitinib, trastuzumab, saracatinib). We observed that these antihormone-induced phenomena are common to ER+HER2- and ER+HER2+ breast cancer models in vitro, where targeting of EGFR, HER2 or Src alongside antihormone improves antitumour response and delays/prevents endocrine resistance. Such targeted inhibitors also subvert acquired endocrine resistant cells which retain increased EGFR, HER2 and Src (e.g., TAMR and FASR models derived after 6-12 months of tamoxifen or Faslodex treatment). Thus, antihormone-induced tyrosine kinases comprise "compensatory signalling" crucial in limiting maximal initial antihormone response and subsequently driving acquired resistance in vitro. However, despite such convincing preclinical findings from our group and others, clinical trials examining equivalent antigrowth factor strategies have proved relatively disappointing. Our new studies deciphering underlying causes reveal that further antihormone-promoted events could be pivotal in vivo. Firstly, Faslodex induces HER3 and HER4 which sensitise ER+ cells to heregulin, a paracrine growth factor that overcomes endocrine response and diminishes antitumour effect of agents targeting EGFR, HER2 or Src alongside antihormone. Secondly, extended antihormone exposure (experienced by ER+ cells prior to adjuvant clinical relapse) can "reprogramme" the compensatory kinase profile in vitro, hindering candidate antigrowth factor targeting of endocrine resistance. Faslodex resistant cells maintained with this antihormone for 3 years in vitro lose EGFR/HER2 dependency, gaining alternative mitogenic/invasion kinases. Deciphering these previously unrecognised antihormone-induced events could provide superior treatments to control endocrine relapse in the clinic.

Growth of hormone-dependent MCF-7 breast cancer cells is promoted by constitutive caveolin-1 whose expression is lost in an EGF-R-mediated manner during development of tamoxifen resistance.

Caveolin-1 displays both tumour-suppressor and tumour-promoter properties in breast cancer. Using characterised preclinical cell models for the transition of oestrogen-sensitive (WT-MCF-7 cells) to a tamoxifen-resistant (TAM-R cells) phenotype we examined the role caveolin-1 in the development of hormone-resistant breast cancer. The WT-MCF-7 cells showed abundant expression of caveolin-1 which potentiated oestrogen-receptor (ERalpha) signalling and promoted cell growth despite caveolin-1 mediating inhibition of ERK signalling. In TAM-R cells caveolin-1 expression was negligible, repressed by EGF-R/ERK signalling. Pharmacological inhibition of EGFR/ERK in TAM-R cells restored caveolin-1 and also resulted in the emergence of pools of phosphorylated caveolin-1. WT-MCF-7 cells exposed to tamoxifen for upto 12 weeks displayed increased caveolin-1 (peaking by week 2) followed (after week 8) by a marked decrease as the cells progress to develop a stable tamoxifen-resistant phenotype. The targeted down-regulation (siRNA) of caveolin-1 in WT-MCF-7 cells reduced growth but did not affect their sensitivity to tamoxifen, suggesting loss of caveolin-1 alone is not sufficient to confer tamoxifen-resistance. Hyperactivation of EGFR/ERK is a feature of tamoxifen-resistant breast cancer cells, a principal driver of cell growth. Recombinant expression of caveolin-1 in TAM-R cells did not affect EGFR/ERK activity, potentially due to mislocalisation of caveolin-1 through hyperactivation of the mTOR pathway or altered caveolin-1 phosphorylation. This work defines a novel role for caveolin-1 with implications for the clinical course of breast cancer and identifies caveolin-1 as a potential drug target for the treatment of early oestrogen-dependent breast cancers. Further, the loss of caveolin-1 may have benefit as a molecular signature for tamoxifen resistance.

Regulation of ERBB2 by oestrogen receptor-PAX2 determines response to tamoxifen.

Crosstalk between the oestrogen receptor (ER) and ERBB2/HER-2 pathways has long been implicated in breast cancer aetiology and drug response, yet no direct connection at a transcriptional level has been shown. Here we show that oestrogen-ER and tamoxifen-ER complexes directly repress ERBB2 transcription by means of a cis-regulatory element within the ERBB2 gene in human cell lines. We implicate the paired box 2 gene product (PAX2), in a previously unrecognized role, as a crucial mediator of ER repression of ERBB2 by the anti-cancer drug tamoxifen. We show that PAX2 and the ER co-activator AIB-1/SRC-3 compete for binding and regulation of ERBB2 transcription, the outcome of which determines tamoxifen response in breast cancer cells. The repression of ERBB2 by ER-PAX2 links these two breast cancer subtypes and suggests that aggressive ERBB2-positive tumours can originate from ER-positive luminal tumours by circumventing this repressive mechanism. These data provide mechanistic insight into the molecular basis of endocrine resistance in breast cancer.

Insulin receptor substrate-1 involvement in epidermal growth factor receptor and insulin-like growth factor receptor signalling: implication for Gefitinib ('Iressa') response and resistance.

Classically the insulin receptor substrate-1 (IRS-1) is an essential component of insulin-like growth factor type 1 receptor (IGF-IR) signalling, providing an interface between the receptor and key downstream signalling cascades. Here, however, we show that in tamoxifen-resistant MCF-7 (Tam-R) breast cancer cells, that are highly dependent on epidermal growth factor receptor (EGFR) for growth, IRS-1 can interact with EGFR and be preferentially phosphorylated on tyrosine (Y) 896, a Grb2 binding site. Indeed, phosphorylation of this site is greatly enhanced by exposure of these cells, and other EGFR-positive cell lines, to EGF. Importantly, while IGF-II promotes phosphorylation of IRS-1 on Y612, a PI3-K recruitment site, it has limited effect on Y896 phosphorylation in Tam-R cells. Furthermore, EGF and IGF-II co-treatment, reduces the ability of IGF-II to phosphorylate Y612, whilst maintaining Y896 phosphorylation, suggesting that the EGFR is the dominant recruiter of IRS-1 in this cell line. Significantly, challenge of Tam-R cells with the EGFR-selective tyrosine kinase inhibitor gefitinib, for 7 days, reduces IRS-1/EGFR association and IRS-1 Y896 phosphorylation, while promoting IRS-1/IGF-IR association and IRS-1 Y612 phosphorylation. Furthermore, gefitinib significantly enhances IGF-II-mediated phosphorylation of IRS-1 Y612 and AKT in Tam-R cells. Importantly, induction of this pathway by gefitinib can be abrogated by inhibition/downregulation of the IGF-IR. Our data would therefore suggest a novel association exists between the EGFR and IRS-1 in several EGFR-positive cancer cell lines. This association acts to promote phosphorylation of IRS-1 at Y896 and drive MAPK signalling whilst preventing recruitment of IRS-1 by the IGF-IR and inhibiting signalling via this receptor. Treatment with gefitinib alters the dynamics of this system, promoting IGF-IR signalling, the dominant gefitinib-resistant growth regulatory pathway in Tam-R cells, thus, potentially limiting its efficacy.

Heregulin beta1 drives gefitinib-resistant growth and invasion in tamoxifen-resistant MCF-7 breast cancer cells.

INTRODUCTION: Resistance to anti-epidermal growth factor receptor (anti-EGFR) therapies is an emerging clinical problem. The efficacy of anti-EGFR therapies can be influenced by the presence of heregulins (HRGs), which can bind erbB3/4 receptors and can activate alternative signalling pathways. In the present study we have examined whether HRG signalling can circumvent EGFR blockade in an EGFR-positive tamoxifen-resistant MCF-7 (Tam-R) breast cancer cell line. METHODS: Tam-R cells, incubated with the selective EGFR tyrosine kinase inhibitor gefitinib ('Iressa', ZD1839), were exposed to HRGbeta1 and the effects on erbB receptor dimerization profiles and on activation of associated downstream signalling components were assessed by immunoprecipitation, western blotting and immunocytochemistry. The effects of HRGbeta1 on gefitinib-treated Tam-R cell growth and invasion were also examined, and HRGbeta1 expression levels were assessed in breast cancer tissue by immunohistochemistry to address the potential clinical relevance of such a resistance mechanism. RESULTS: In Tam-R cells, HRGbeta1 promoted erbB3/erbB2 and erbB3/EGFR heterodimerization, promoted ERK1/2 and AKT pathway activation and increased cell proliferation and invasion. Gefitinib prevented HRGbeta1-driven erbB3/EGFR heterodimerization, ERK1/2 activation and Tam-R cell proliferation, but HRGbeta1-driven erbB3/erbB2 heterodimerization, AKT activation and Tam-R cell invasion were maintained. A combination of gefitinib and the phosphatidylinositol 3-kinase inhibitor LY294002 effectively blocked HRGbeta1-mediated intracellular signalling activity, growth and invasion in Tam-R cells. Similarly, targeting erbB2 with trastuzumab in combination with gefitinib in Tam-R cells reduced HRGbeta1-induced erbB2 and ERK1/2 activity; however, HRGbeta1-driven AKT activity and cell growth were maintained while cell invasion was significantly enhanced with this combination. In clinical tissue all samples demonstrated cytoplasmic tumour epithelial HRGbeta1 protein staining, with expression correlating with EGFR positivity and activation of both AKT and ERK1/2. CONCLUSION: HRGbeta1 can overcome the inhibitory effects of gefitinib on cell growth and invasion in Tam-R cells through promotion of erbB3/erbB2 heterodimerization and activation of the phosphatidylinositol 3-kinase/AKT signalling pathway. This may have implications for the effectiveness of anti-EGFR therapies in breast cancer as HRGbeta1 is enriched in many EGFR-positive breast tumours.

Growth factor receptor interplay and resistance in cancer.

Aberrant signalling through the epidermal growth factor receptor (EGFR) plays a major role in the progression and maintenance of the malignant phenotype and the receptor is therefore a rational anti-cancer target. A variety of approaches have been developed to specifically target the EGFR which include monoclonal antibodies and small molecule tyrosine kinase inhibitors, such as gefitinib (Iressa). However, the recent clinical experience across a range of cancer types is revealing that despite the anti-EGFR agents demonstrating some anti-tumour activity, there is a high level of de novo and acquired resistance to such treatments and moreover, overexpression of the EGFR is clearly not the sole determinant of response to such therapies. Such adverse phenomena, which serve to limit the overall therapeutic impact of these new agents, implies the existence of a greater complexity involved in the regulation of EGFR signalling than was previously assumed. Indeed, evidence is accumulating which demonstrates that signalling interplay occurs between the EGFR, and the IGF-1 receptor (IGF-1R) and the review will focus on the emerging concept of growth factor pathway switching between these two receptors as a means of influencing the effectiveness of anti-EGFR agents such as gefitinib.

Inductive mechanisms limiting response to anti-epidermal growth factor receptor therapy.

Aberrant epidermal growth factor receptor (EGFR) signalling, a key feature of a variety of human malignancies, can drive a range of mechanisms underlying tumour growth and progression, including increased cell proliferation, angiogenesis, metastasis and decreased apoptosis. Anti-EGFR therapies, as monotherapies and in combination with chemotherapy, have proved effective in inhibiting these processes both in the clinical and in the preclinical settings. However, only a small cohort of patients have derived significant benefit from this therapy, with both de novo and acquired resistance to these agents evident in a number of recent studies. If we are to improve the effectiveness of such targeted therapies, then there is an urgent need to understand the resistance mechanisms. Here, we describe both non-genomic and genomic mechanisms of resistance to the selective EGFR tyrosine kinase inhibitor gefitinib (IRESSA), which we have identified initially in an EGFR-positive tamoxifen-resistant MCF-7 breast cancer cell line, but more recently in other EGFR-positive cancer types. Importantly, we show that gefitinib, in common with anti-hormonal agents, is not a passive bystander in the cellular response to drug treatment, but plays an active role in promoting signalling pathways that serve to limit its anti-tumour activity and maintain the cellular cohort from which acquired resistance can ultimately evolve. These findings indicate that inductive signalling is an important determinant of response to EGFR-targeted therapies and deciphering such pathways may provide us with the opportunity to design more effective strategies to combat resistance mechanisms and improve response to initial therapy.

Insulin-like growth factor-I receptor signaling and resistance in breast cancer.

Insulin-like growth factor-I receptor (IGF-IR) signaling is involved in many fundamental adverse aspects of cancer cell biology, such as proliferation, cell survival and migration. Its anti-apoptotic properties have implicated the receptor in mediating decreased sensitivity to chemotherapeutic drugs and radiation treatment; however, data are emerging that also indicates a role for IGF-IR signaling in resistance, not only to antihormones but also to antigrowth factor strategies such as agents that target the erb family of receptors. As such, IGF-IR is clearly an attractive therapeutic target for the treatment of cancer, including breast cancer, where there is evidence of clinical prominence of the IGF-IR pathway and, as such, numerous strategies are currently in development to inhibit IGF-IR signaling. This review focuses on the ability of the IGF-IR to contribute to resistance mechanisms that support breast cancer cell growth in the presence of antihormones and antigrowth factors and discusses methods to maximize antitumor effects by combination regimens cotargeting the IGF-IR that may delay, or even prevent, progression to the resistant phenotype.

Nonendocrine pathways and endocrine resistance: observations with antiestrogens and signal transduction inhibitors in combination.

An increasing body of evidence demonstrates that growth factor networks are highly interactive with estrogen receptor signaling in the control of breast cancer growth. As such, tumor responses to antiestrogens are likely to be a composite of the estrogen receptor and growth factor-inhibitory activity of these agents, with alterations/aberrations in growth factor signaling providing a mechanism for the development of antiestrogen resistance. In this light, the current article focuses on illustrating the relationship between growth factor signaling and antiestrogen failure in our in-house tumor models of breast cancer and describing how we are now beginning to successfully target growth factor activity to improve the effects of antiestrogen drugs and to block aggressive disease progression.

The biology of antihormone failure in breast cancer.

Many estrogen receptor-positive breast cancer patients initially respond to treatment with antihormonal agents such as tamoxifen, but remissions are often followed by acquisition of resistance and ultimately disease relapse. The development of a rationale for the effective treatment of tamoxifen-resistant breast cancer requires an understanding of the complex signal transduction mechanisms that contribute towards loss of antiestrogen response. Interactions between estrogen and growth factor signaling pathways have been identified in estrogen-responsive cells that are thought to reinforce their individual cellular effects on growth and gene responses. Increasing evidence indicates that abnormalities occurring in growth factor signaling pathways, notably the epidermal growth factor receptor (EGFR) signaling pathway, could dramatically influence steroid hormone action and may be critical to antihormonal-resistant breast cancer cell growth. Thus, inhibitory agents targeting growth factor receptors, or their intracellular pathway components, may prove clinically beneficial in antihormone refractory disease. One example, gefitinib ('Iressa', ZD1839), an EGFR-tyrosine kinase inhibitor, is an interesting therapeutic option that may provide benefit in the treatment of antihormonal-resistant breast cancer. Rapid progress with pharmacological and molecular therapeutic agents is now being made. Therapies that target growth factor signaling pathways may prevent development of resistance.

Oestrogen receptor-mediated modulation of the EGFR/MAPK pathway in tamoxifen-resistant MCF-7 cells.

Oestrogen receptor (ER) levels are usually maintained on acquisition of tamoxifen resistance in the clinic, however, tumour re-growth is associated with increased expression of epidermal growth factor receptor (EGFR) and activation of the mitogen activated protein kinase (MAPK) pathway. In the present study we have used the ER down-regulator fulvestrant ('Faslodex') to investigate the influence of the ER on growth of a tamoxifen-resistant (TAM-R) human breast cancer cell line. Expression levels of ER mRNA and protein were equivalent in parental wild-type MCF-7 (WT) and TAM-R cells. Fulvestrant eliminated ER protein expression and inhibited proliferation in both cell lines. The growth inhibitory effects of fulvestrant were associated with a decrease in basal EGFR, c-erbB2 and ERK1/2 activity in TAM-R but not WT cells. ER functionality as determined by oestrogen response element (ERE)-luciferase reporter activity and expression of PgR, pS2 and transforming growth factor alpha (TGFalpha) was significantly reduced in TAM-R compared to WT cells and was further decreased by fulvestrant treatment in both cell lines. Epidermal growth factor (EGF) and TGFalpha significantly increased EGFR/MAPK pathway activity in both cell lines. Ligand-induced EGFR/MAPK activation promoted TAM-R cell growth in both the absence and presence of fulvestrant, whereas no proliferative activity was observed under the same conditions in WT cells. These results suggest that the ER modulates EGFR/MAPK signalling efficiency in TAM-R cells possibly through the regulation of TGFalpha availability. This effect may be overcome by the action of exogenous EGFR ligands, which strengthen EGFR/MAPK signalling activity to generate endocrine-insensitive cell growth.

Elevated levels of epidermal growth factor receptor/c-erbB2 heterodimers mediate an autocrine growth regulatory pathway in tamoxifen-resistant MCF-7 cells.

The development of acquired resistance to antihormonal agents in breast cancer is a major therapeutic problem. We have developed a tamoxifen-resistant (TAM-R) MCF-7 breast cancer cell line to investigate the mechanisms behind this condition. Both epidermal growth factor receptor (EGFR) and c-erbB2 mRNA and protein expression were increased in TAM-R compared with wild-type MCF-7 cells, whereas comparable levels of c-erbB3 mRNA and protein were expressed in both cell lines. Under basal conditions, phosphorylated EGFR/c-erbB2, EGFR/c-erbB3 but not c-erbB2/c-erbB3 receptor heterodimers were detected in TAM-R cells in association with increased levels of phosphorylated extracellular-signal regulated kinase 1/2 (ERK1/2). Both cell lines were capable of generating a range of EGFR-specific ligands and increased expression of transforming growth factor alpha was observed in TAM-R cells. Treatment of TAM-R cells with ZD1839 (Iressa) or trastuzumab (Herceptin) blocked c-erbB receptor heterodimer formation and phosphorylation, reduced ERK1/2 activity, and strongly inhibited cell growth. The MAPK kinase inhibitor PD098059 specifically reduced phosphorylated ERK1/2 levels and inhibited TAM-R growth. All three agents abolished ERK1/2 activity in wild-type cells but caused only small reductions in cell proliferation. These results demonstrate that TAM-R MCF-7 cell growth is mediated by the autocrine release and action of an EGFR-specific ligand inducing preferential EGFR/c-erbB2 dimerization and downstream activation of the ERK pathway.