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.

Biological effects of fulvestrant on estrogen receptor positive human breast cancer: short, medium and long-term effects based on sequential biopsies.

We report the first study of the biological effect of fulvestrant on ER positive clinical breast cancer using sequential biopsies through to progression. Thirty-two locally/systemically advanced breast cancers treated with first-line fulvestrant (250 mg/month) were biopsied at therapy initiation, 6 weeks, 6 months and progression and immunohistochemically-analyzed for Ki67, ER, EGFR and HER2 expression/signaling activity. This series showed good fulvestrant responses (duration of response [DoR] = 25.8 months; clinical benefit = 81%). Ki67 fell (p < 0.001) in 79% of tumours by 6 months and lower Ki67 at all preprogression time-points predicted for longer DoR. ER and PR significantly decreased in all tumours by 6 months (p < 0.001), with some declines in ER (serine 118) phosphorylation and Bcl-2 (p = 0.007). There were modest HER2 increases (p = 0.034, 29% tumours) and loss of any detectable EGFR phosphorylation (p = 0.024, 50% tumours) and MAP kinase (ERK1/2) phosphorylation (p = 0.019, 65% tumours) by 6 months. While ER remained low, there was some recovery of Ki67, Bcl-2 and (weakly) EGFR/MAPK activity in 45-67% patients at progression. Fulvestrant's anti-proliferative impact is related to DoR, but while commonly downregulating ER and indicators of its signaling and depleting EGFR/MAPK signaling in some patients, additional elements must determine response duration. Residual ER at fulvestrant relapse explains reported sensitivity to further endocrine therapies. Occasional modest treatment-induced HER2 and weakly detectable EGFR/HER2/MAPK signaling at relapse suggests targeting of such activity might have value alongside fulvestrant in some patients. However, unknown pathways must drive relapse in most. Ki67 has biomarker potential to predict fulvestrant outcome and as a quantitative measure of response.

ELF5 suppresses estrogen sensitivity and underpins the acquisition of antiestrogen resistance in luminal breast cancer.

We have previously shown that during pregnancy the E-twenty-six (ETS) transcription factor ELF5 directs the differentiation of mammary progenitor cells toward the estrogen receptor (ER)-negative and milk producing cell lineage, raising the possibility that ELF5 may suppress the estrogen sensitivity of breast cancers. To test this we constructed inducible models of ELF5 expression in ER positive luminal breast cancer cells and interrogated them using transcript profiling and chromatin immunoprecipitation of DNA followed by DNA sequencing (ChIP-Seq). ELF5 suppressed ER and FOXA1 expression and broadly suppressed ER-driven patterns of gene expression including sets of genes distinguishing the luminal molecular subtype. Direct transcriptional targets of ELF5, which included FOXA1, EGFR, and MYC, accurately classified a large cohort of breast cancers into their intrinsic molecular subtypes, predicted ER status with high precision, and defined groups with differential prognosis. Knockdown of ELF5 in basal breast cancer cell lines suppressed basal patterns of gene expression and produced a shift in molecular subtype toward the claudin-low and normal-like groups. Luminal breast cancer cells that acquired resistance to the antiestrogen Tamoxifen showed greatly elevated levels of ELF5 and its transcriptional signature, and became dependent on ELF5 for proliferation, compared to the parental cells. Thus ELF5 provides a key transcriptional determinant of breast cancer molecular subtype by suppression of estrogen sensitivity in luminal breast cancer cells and promotion of basal characteristics in basal breast cancer cells, an action that may be utilised to acquire antiestrogen resistance.

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.

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.

A randomized trial to assess the biological activity of short-term (pre-surgical) fulvestrant 500 mg plus anastrozole versus fulvestrant 500 mg alone or anastrozole alone on primary breast cancer.

INTRODUCTION: Fulvestrant shows dose-dependent biological activity. Greater estrogen-receptor (ER) blockade may feasibly be achieved by combining fulvestrant with anastrozole. This pre-surgical study compared fulvestrant plus anastrozole versus either agent alone in patients with ER-positive breast cancer. METHODS: In this double-blind, multicenter trial, 121 patients received fulvestrant 500 mg on Day 1 plus anastrozole 1 mg/day for 14 to 21 days (F + A); fulvestrant plus anastrozole placebo (F); or fulvestrant placebo plus anastrozole (A), 2 to 3 weeks before surgery. ER, progesterone-receptor (PgR) and Ki67 expression were determined from tumor biopsies before treatment and at surgery. RESULTS: A total of 103 paired samples were available (F, n = 35; F+A, n = 31; A, n = 37). All treatments significantly reduced mean ER expression from baseline (F: -41%, P = 0.0001; F + A: -39%, P = 0.0001; A: -13%, P = 0.0034). F and F + A led to greater reductions in ER versus A (both P = 0.0001); F + A did not lead to additional reductions versus F. PgR and Ki67 expression were significantly reduced with all treatments (means were -34% to -45%, and -75% to -85%, respectively; all P = 0.0001), with no differences between groups. CONCLUSIONS: In this short-term study, all treatments reduced ER expression, although F and F + A showed greater reductions than A. No significant differences were detected between the treatment groups in terms of PgR and Ki67 expression. No additional reduction in tumor biomarkers with combination treatment was observed, suggesting that F + A is unlikely to have further clinical benefit over F alone. TRIAL REGISTRATION: Clinicaltrials.gov NCT00259090.

Dose-dependent change in biomarkers during neoadjuvant endocrine therapy with fulvestrant: results from NEWEST, a randomized Phase II study.

NEWEST (Neoadjuvant Endocrine Therapy for Women with Estrogen-Sensitive Tumors) is the first study to compare biological and clinical activity of fulvestrant 500 versus 250 mg in the neoadjuvant breast cancer setting. We hypothesized that fulvestrant 500 mg may be superior to 250 mg in blocking estrogen receptor (ER) signaling and growth. A multicenter, randomized, open-label, Phase II study was performed to compare fulvestrant 500 mg (500 mg/month plus 500 mg on day 14 of month 1) versus fulvestrant 250 mg/month for 16 weeks prior to surgery in postmenopausal women with ER+ locally advanced breast cancer. Core biopsies at baseline, week 4, and surgery were assessed for biomarker changes. Primary endpoint: change in Ki67 labeling index (LI) from baseline to week 4 determined by automated computer imaging system (ACIS). Secondary endpoints: ER protein expression and function; progesterone receptor (PgR) expression; tumor response; tolerability. ER and PgR were examined retrospectively using the H score method. A total of 211 patients were randomized (fulvestrant 500 mg: n = 109; 250 mg: n = 102). At week 4, fulvestrant 500 mg resulted in greater reduction of Ki67 LI and ER expression versus 250 mg (-78.8 vs. -47.4% [p < 0.0001] and -25.0 vs. -13.5% [p = 0.0002], respectively [ACIS]); PgR suppression was not significantly different (-22.7 vs. -17.6; p = 0.5677). However, H score detected even greater suppression of ER (-50.3 vs. -13.7%; p < 0.0001) and greater PgR suppression (-80.5 vs. -46.3%; p = 0.0018) for fulvestrant 500 versus 250 mg. At week 16, tumor response rates were 22.9 and 20.6% for fulvestrant 500 and 250 mg, respectively, with considerable decline in all markers by both ACIS and H score. No detrimental effects on endometrial thickness or bone markers and no new safety concerns were identified. This provides the first evidence of greater biological activity for fulvestrant 500 versus 250 mg in depleting ER expression, function, and growth.

A review of the biological and clinical characteristics of luminal-like oestrogen receptor-positive breast cancer.

Global gene expression profiling (GEP) studies of breast cancer have identified distinct biological classes with different clinical and therapeutic implications. Oestrogen receptor (ER) has been found to be a central marker of the molecular signature. GEP studies have consistently recognized a molecularly distinct class of tumours that is characterized by high-level expression of ER and other biomarkers recognized to be characteristic of normal luminal cells of the breast. This class is the largest of the GEP-defined molecular subclasses, comprising 60-70% of breast cancer cases. Moreover, it has been proposed that this group of tumours is composed of at least two subclasses distinguished by differing GEP profiles. At present, there is no consensus on the definition of the luminal subclasses and, in clinical practice, luminal-like tumours and ER-positive tumours are frequently considered to be the same. A better understanding of the biological features of luminal tumours could lead to their improved characterization and consistent identification. In this review, we explore the concept and definitions of the luminal-like class of breast carcinoma and their contribution to our understanding of their molecular features, clinical significance and therapeutic implications.

Overexpression of CD44 accompanies acquired tamoxifen resistance in MCF7 cells and augments their sensitivity to the stromal factors, heregulin and hyaluronan.

BACKGROUND: Acquired resistance to endocrine therapy in breast cancer is a significant problem with relapse being associated with local and/or regional recurrence and frequent distant metastases. Breast cancer cell models reveal that endocrine resistance is accompanied by a gain in aggressive behaviour driven in part through altered growth factor receptor signalling, particularly involving erbB family receptors. Recently we identified that CD44, a transmembrane cell adhesion receptor known to interact with growth factor receptors, is upregulated in tamoxifen-resistant (TamR) MCF7 breast cancer cells. The purpose of this study was to explore the consequences of CD44 upregulation in an MCF7 cell model of acquired tamoxifen resistance, specifically with respect to the hypothesis that CD44 may influence erbB activity to promote an adverse phenotype. METHODS: CD44 expression in MCF7 and TamR cells was assessed by RT-PCR, Western blotting and immunocytochemistry. Immunofluorescence and immunoprecipitation studies revealed CD44-erbB associations. TamR cells (± siRNA-mediated CD44 suppression) or MCF7 cells (± transfection with the CD44 gene) were treated with the CD44 ligand, hyaluronon (HA), or heregulin and their in vitro growth (MTT), migration (Boyden chamber and wound healing) and invasion (Matrigel transwell migration) determined. erbB signalling was assessed using Western blotting. The effect of HA on erbB family dimerisation in TamR cells was determined by immunoprecipitation in the presence or absence of CD44 siRNA. RESULTS: TamR cells overexpressed CD44 where it was seen to associate with erbB2 at the cell surface. siRNA-mediated suppression of CD44 in TamR cells significantly attenuated their response to heregulin, inhibiting heregulin-induced cell migration and invasion. Furthermore, TamR cells exhibited enhanced sensitivity to HA, with HA treatment resulting in modulation of erbB dimerisation, ligand-independent activation of erbB2 and EGFR and induction of cell migration. Overexpression of CD44 in MCF7 cells, which lack endogenous CD44, generated an HA-sensitive phenotype, with HA-stimulation promoting erbB/EGFR activation and migration. CONCLUSIONS: These data suggest an important role for CD44 in the context of tamoxifen-resistance where it may augment cellular response to erbB ligands and HA, factors that are reported to be present within the tumour microenvironment in vivo. Thus CD44 may present an important determinant of breast cancer progression in the setting of endocrine resistance.

Pro-metastatic tumor-stroma interactions in breast cancer.

The vast majority of breast cancer-related deaths are due to metastatic disease. Reciprocal and complex interactions between epithelial tumor cells and the various components of the tumor microenvironment influence tumor progression and metastases although the molecular mechanisms underlying these metastasis-promoting effects are not fully characterized. Identifying and understanding pathways of tumor-stroma cross-talk are likely to lead to the development of novel prognostic biomarkers for metastasis and strategies to prevent metastasis at its earliest stages, resulting in improved patient outcomes.

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.

Inhibition of focal adhesion kinase suppresses the adverse phenotype of endocrine-resistant breast cancer cells and improves endocrine response in endocrine-sensitive cells.

Acquired resistance to endocrine therapy in breast cancer is a major clinical problem. Previous reports have demonstrated that cell models of acquired endocrine resistance have altered cell-matrix adhesion and a highly migratory phenotype, features which may impact on tumour spread in vivo. Focal adhesion kinase (FAK) is an intracellular kinase that regulates signalling pathways central to cell adhesion, migration and survival and its expression is frequently deregulated in breast cancer. In this study, we have used the novel FAK inhibitor PF573228 to address the role of FAK in the development of endocrine resistance. Whilst total-FAK expression was similar between endocrine-sensitive and endocrine-resistant MCF7 cells, FAK phosphorylation status (Y397 or Y861) was altered in resistance. PF573228 promoted a dose-dependent inhibition of FAK phosphorylation at Y397 but did not affect other FAK activation sites (pY407, pY576 and pY861). Endocrine-resistant cells were more sensitive to these inhibitory effects versus MCF7 (mean IC(50) for FAK pY397 inhibition: 0.43 µM, 0.05 µM and 0.13 µM for MCF7, TamR and FasR cells, respectively). Inhibition of FAK pY397 was associated with a reduction in TamR and FasR adhesion to, and migration over, matrix components. PF573228 as a single agent (0-1 µM) did not affect the growth of MCF7 cells or their endocrine-resistant counterparts. However, treatment of endocrine-sensitive cells with PF573228 and tamoxifen combined resulted in greater suppression of proliferation versus single agent treatment. Together these data suggest the importance of FAK in the process of endocrine resistance, particularly in the development of an aggressive, migratory cell phenotype and demonstrate the potential to improve endocrine response through combination treatment.

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.

Transferrin receptor (CD71) is a marker of poor prognosis in breast cancer and can predict response to tamoxifen.

Transferrin receptor (CD71) is involved in the cellular uptake of iron and is expressed on cells with high proliferation. It may be implicated in promoting the growth of endocrine resistant phenotypes within ER+/luminal-like breast cancer. We used a panel of in vitro cell models of acquired resistance to tamoxifen (TAMR), Faslodex (FASR) or severe oestrogen deprivation (MCF-7X) and the ER+ luminal MCF-7 parental line to determine CD71 mRNA expression and to study transferrin (Tf) effects on in vitro tumour growth and its inhibition. Furthermore, CD71 protein expression was assessed in a well-characterized series of patients with invasive breast carcinoma using tissue microarrays. Our results demonstrated a striking elevation of CD71 in all cell models of acquired resistance. Exogenous Tf significantly promoted growth in MCF-7-X and MCF-7 cells but more so in MCF-7-X; this growth was significantly reduced by Faslodex (FAS) or a phosphoinositide-3 kinase inhibitor (LY294002). Increased CD71 expression was associated with poor NPI score, tumour proliferation, basal CKs, p53, EGFR, HER2, steroid receptor negativity and shortened breast cancer specific survival (P < 0.001). On multivariate analysis, CD71 was found to be an independent prognostic factor in the ER+ cohort of patients. In conclusion, therapies of current interest in breast cancer (e.g. FAS, PI3K-inhibitors) appear able to partially impact on transferrin/CD71-promoted growth, but further investigation of this important mitogenic mechanism may assist in designing new therapeutic strategies to target highly proliferative, endocrine resistant breast cancers. CD71 appears to be a candidate marker of a subgroup of ER+/luminal-like breast cancer characterised by poor outcome and resistance to tamoxifen.

Expression of a phosphorylated p130(Cas) substrate domain attenuates the phosphatidylinositol 3-kinase/Akt survival pathway in tamoxifen resistant breast cancer cells.

Elevated expression of p130(Cas)/BCAR1 (breast cancer anti estrogen resistance 1) in human breast tumors is a marker of poor prognosis and poor overall survival. Specifically, p130(Cas) signaling has been associated with antiestrogen resistance, for which the mechanism is currently unknown. TAM-R cells, which were established by long-term exposure of estrogen (E(2))-dependent MCF-7 cells to tamoxifen, displayed elevated levels of total and activated p130(Cas). Here we have investigated the effects of p130(Cas) inhibition on growth factor signaling in tamoxifen resistance. To inhibit p130(Cas), a phosphorylated substrate domain of p130(Cas), that acts as a dominant-negative (DN) p130(Cas) molecule by blocking signal transduction downstream of the p130(Cas) substrate domain, as well as knockdown by siRNA was employed. Interference with p130(Cas) signaling/expression induced morphological changes, which were consistent with a more epithelial-like phenotype. The phenotypic reversion was accompanied by reduced migration, attenuation of the ERK and phosphatidylinositol 3-kinase/Akt pathways, and induction of apoptosis. Apoptosis was accompanied by downregulation of the expression of the anti-apoptotic protein Bcl-2. Importantly, these changes re-sensitized TAM-R cells to tamoxifen treatment by inducing cell death. Therefore, our findings suggest that targeting the product of the BCAR1 gene by a peptide which mimics the phosphorylated substrate domain may provide a new molecular avenue for treatment of antiestrogen resistant breast cancers.

ZIP7-mediated intracellular zinc transport contributes to aberrant growth factor signaling in antihormone-resistant breast cancer Cells.

Antiestrogens such as tamoxifen are the mainstay of treatment for estrogen receptor-positive breast cancer. However, their effectiveness is limited by the development of endocrine resistance, allowing tumor regrowth and progression. Importantly, in vitro MCF7 cell models of acquired tamoxifen resistance (TamR cells) display an aggressive, invasive phenotype in which activation of epithelial growth factor receptor/IGF-I receptor/Src signaling plays a critical role. In this study, we report that TamR cells have increased levels of zinc and zinc transporter, ZIP7 [solute carrier family 39 (zinc transporter) member 7, also known as SLC39A7], resulting in an enhanced response to exogenous zinc, which is manifested as a greatly increased growth factor receptor activation, leading to increased growth and invasion. Removal of ZIP7, using small interfering RNA, destroys this activation of epithelial growth factor receptor/IGF-I receptor/Src signaling by reducing intracellular zinc levels. Similarly, it also blocks the activation of HER2, -3, and -4. These data suggest that intracellular zinc levels may be a critical factor in determining growth factor responses and that the targeting of zinc transporters may have novel therapeutic implications. We show that ZIP7 is a critical component in the redistribution of zinc from intracellular stores to the cytoplasm and, as such, is essential for the zinc-induced inhibition of phosphatases, which leads to activation of growth factor receptors. Removal of ZIP7 therefore offers a means through which zinc-induced activation of growth factor receptors may be effectively suppressed and provides a mechanism of targeting multiple growth factor pathways, increasing tumor kill, and preventing further development of resistance in breast cancer.

Anti-oestrogens but not oestrogen deprivation promote cellular invasion in intercellular adhesion-deficient breast cancer cells.

INTRODUCTION: Anti-oestrogens have been the mainstay of therapy in patients with oestrogen-receptor (ER) positive breast cancer and have provided significant improvements in survival. However, their benefits are limited by tumour recurrence in a significant proportion of initially drug-responsive breast cancer patients because of acquired anti-oestrogen resistance. Relapse on such therapies clinically presents as local and/or regional recurrences, frequently with distant metastases, and the prognosis for these patients is poor. The selective ER modulator, tamoxifen, classically exerts gene inhibitory effects during the drug-responsive phase in ER-positive breast cancer cells. Paradoxically, this drug is also able to induce the expression of genes, which in the appropriate cell context may contribute to an adverse cell phenotype. Here we have investigated the effects of tamoxifen and fulvestrant treatment on invasive signalling and compared this with the direct effects of oestrogen withdrawal to mimic the action of aromatase inhibitors. METHODS: The effect of oestrogen and 4-hydroxy-tamoxifen on the invasive capacity of endocrine-sensitive MCF-7 cells, in the presence or absence of functional E-cadherin, was determined by Matrigel invasion assays. Studies also monitored the impact of oestrogen withdrawal or treatment with fulvestrant on cell invasion. Western blotting using phospho-specific antibodies was performed to ascertain changes in invasive signalling in response to the two anti-oestrogens versus both oestradiol treatment and withdrawal. RESULTS: To the best of our knowledge, we report for the first time that tamoxifen can promote an invasive phenotype in ER-positive breast cancer cells under conditions of poor cell-cell contact and suggest a role for Src kinase and associated pro-invasive genes in this process. Our studies revealed that although this adverse effect is also apparent for further classes of anti-oestrogens, exemplified by the steroidal agent fulvestrant, it is absent during oestrogen withdrawal. CONCLUSIONS: These data highlight a previously unreported effect of tamoxifen (and potentially further anti-oestrogens), that such agents appear able to induce breast cancer cell invasion in a specific context (absence of good cell-cell contacts), where these findings may have major clinical implications for those patients with tumours that have inherently poor intercellular adhesion. In such patients oestrogen deprivation with aromatase inhibitors may be more appropriate.

Forkhead-box A1 (FOXA1) expression in breast cancer and its prognostic significance.

The forkhead-box A1 (FOXA1) controls downstream transcription of oestrogen receptor (ER)-regulated genes. In this study, the biological and prognostic value of FOXA1 expression was assessed immunohistochemically in a large and well-characterised series of invasive breast carcinoma with a long term follow-up using tissue microarray. FOXA1 expression was associated with steroid hormone receptors (ERalpha, PgR and AR), other variables of good prognosis such as smaller tumour size, lower histological grade, luminal cytokeratins (CK18 and CK7/8), BRCA1 and E-cadherin. Its expression showed an inverse relation with basal CKs (CK14 and CK5/6) and P-cadherin. We found an association between high FOXA1 expression and a better survival in the whole series however; multivariate analysis showed that FOXA1 was not an independent prognostic marker. In conclusion, our results show that FOXA1 protein is associated with markers of good prognosis supporting its role as a growth repressor in breast cancer. In this series, FOXA1 was found not to be of an independent prognostic significance in breast cancer and as such its immunohistochemical assessment alone does not appear to have relevance in routine practice to stratify ER-positive (luminal-like) tumours into clinically significant subgroups.

Invasive lobular carcinoma of the breast: response to hormonal therapy and outcomes.

UNLABELLED: Invasive lobular carcinoma (ILC) comprises approximately 5-15% of breast cancers and appears to have a distinct biology. It is less common than invasive ductal carcinoma (IDC) and few large studies have addressed its biologic characteristics and behaviour with respect to long-term clinical outcome and response to adjuvant therapy. METHODS: This study is based on a large and well-characterised consecutive series of invasive breast carcinomas with a long-term follow-up (up to 25 years). This series included 415 (8%) patients with pure ILC and 2901 (55.7%) with IDC (not otherwise specified) identified from a consecutive cohort of 5680 breast tumours presented to our Breast Unit that were treated in a similar conventional manner. Clinicopathological, therapy and outcome information as well as data on a large panel of biomarkers were available. RESULTS: Compared to IDC, patients with ILC tended to be older and present with tumours which are more frequently lower grade (typically, grade 2 [84%]), hormone-receptor positive (86% compared to 61% in IDC), of larger size, and with the absence of vascular invasion. A higher frequency of ILC was placed in the good Nottingham Prognostic Index group (40% compared to 21% in IDC). ILC showed indolent but progressive behavioural characteristics with nearly linear survival curves which crossed those of IDC after approximately 10years of follow-up, thus eventually exhibiting a worse long-term outcome. Importantly, ILC showed a better response to adjuvant hormonal therapy (HT) with improvement in survival in patients who received HT compared with matched patients with IDC. CONCLUSION: ILC is a distinct entity of breast cancer that responds well to adjuvant HT. These data add important clinical information for assessing the long-term benefits of adjuvant HT use in ILC.