Denise Barrow

Elevated Src activity promotes cellular invasion and motility in tamoxifen resistant breast cancer cells.

SummarySrc kinase plays a central role in growth factor signalling, regulating a diverse array of cellular functions including proliferation, migration and invasion. Recent studies have demonstrated that Src activity is frequently elevated in human tumours and correlates with disease stage. We have previously demonstrated that, upon acquisition of tamoxifen resistance, MCF7 cells display increased epidermal growth factor receptor (EGFR) activation and a more aggressive phenotype in vitro. Since tumours exhibiting elevated EGFR signalling may possess elevated levels of Src activity, we wished to investigate the role of Src in our MCF7 model of endocrine resistance. Src kinase activity was significantly elevated in tamoxifen-resistant (TamR) cells in comparison to wild type MCF7 cells. This increase was not due to elevated Src protein or gene expression. Treatment of TamR cells with the novel Src inhibitor, AZD0530, significantly reduced the amount of activated Src detectable in both cell types whilst having no effect on total Src levels. AZD0530 significantly suppressed the motile and invasive nature of TamR cells in vitro, reduced basal levels of activated focal adhesion kinase (FAK) and paxillin and promoted elongation of focal adhesions. Furthermore, the use of this compound in conjunction with the EGFR inhibitor, gefitinib, was markedly additive towards inhibition of TamR cell motility and invasion. These observations suggest that Src plays a pivotal role in mediating the motile and invasive phenotype observed in endocrine-resistant breast cancer cells. The use of Src inhibitors in conjunction with EGFR inhibitors such as gefitinib may provide an effective method with which to prevent cancer progression and metastasis.

Tamoxifen resistance in MCF7 cells promotes EMT-like behaviour and involves modulation of β-catenin phosphorylation

We have previously demonstrated that, following acquisition of endocrine resistance, breast cancer cells display an altered growth rate together with increased aggressive behaviour in vitro. Since dysfunctional cell–cell adhesive interactions can promote an aggressive phenotype, we investigated the integrity of this protein complex in our breast cancer model of tamoxifen resistance. In culture, tamoxifen‐resistant MCF7 (TamR) cells grew as loosely packed colonies with loss of cell–cell junctions and demonstrated altered morphology characteristic of cells undergoing epithelial‐to‐mesenchymal transition (EMT). Neutralising E‐cadherin function promoted the invasion and inhibited the aggregation of endocrine‐sensitive MCF7 cells, whilst having little effect on the behaviour of TamR cells. Additionally, TamR cells had increased levels of tyrosine‐phosphorylated β‐catenin, whilst serine/threonine‐phosphorylated β‐catenin was decreased. These cells also displayed loss of association between β‐catenin and E‐cadherin, increased cytoplasmic and nuclear β‐catenin and elevated transcription of β‐catenin target genes known to be involved in tumour progression and EMT. Inhibition of EGFR kinase activity in TamR cells reduced β‐catenin tyrosine phosphorylation, increased β‐catenin–E‐cadherin association and promoted cell–cell adhesion. In such treated cells, the association of β‐catenin with Lef‐1 and the transcription of c‐myc, cyclin‐D1, CD44 and COX‐2 were also reduced. These results suggest that homotypic adhesion in tamoxifen‐resistant breast cancer cells is dysfunctional due to EGFR‐driven modulation of the phosphorylation status of β‐catenin and may contribute to an enhanced aggressive phenotype and transition towards a mesenchymal phenotype in vitro.

Bidirectional cross talk between ERα and EGFR signalling pathways regulates tamoxifen-resistant growth

SummaryWe have previously demonstrated that oestrogen receptor α (ERα) modulates epidermal growth factor receptor (EGFR)/mitogen-activated protein kinase (MAPK) signalling efficiency in a tamoxifen-resistant MCF-7 breast cancer cell line (Tam-R). In the present study we have investigated whether this cross-talk between EGFR/MAPK and ERα signalling pathways is bidirectional by examining the effects of EGFR/MAPK activity on ER functionality in the same cell line. Elevated expression levels of phosphorylated serine 118 (S118) ERα were observed in the Tam-R compared to the parental wild type MCF-7 cell line (WT-MCF-7) under basal growth conditions. Phosphorylation of ERα at S118 was regulated by the EGFR/MAPK pathway in Tam-R cells being increased in response to amphiregulin (AR) and inhibited by the selective EGFR tyrosine kinase inhibitor, gefitinib and the MEK1/2 inhibitor, PD184352. Recruitment of the co-activators p68 RNA helicase and SRC1 to ERα, oestrogen response element (ERE) activity and Tam-R cell growth were similarly EGFR/MAPK-regulated. Chromatin immunoprecipitation (ChIP) studies revealed that in Tam-R cells the ERα assembled on the AR gene promoter and this was associated with elevated basal expression of AR mRNA. Furthermore, AR mRNA expression was under the regulation of the EGFR/MAPK and ERα signalling pathways. Neutralising antibodies to AR inhibited EGFR/ERK1/2 activity, reduced S118 ERα phosphorylation and reduced AR mRNA expression in TAM-R cells. These findings suggest that ERα function in Tam-R cells is maintained as a consequence of EGFR/MAPK-mediated phosphorylation at serine residue 118 resulting in the generation of a self-propogating autocrine growth-regulatory loop through the ERα-mediated production of AR.

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.

Modulation of Epidermal Growth Factor Receptor in Endocrine‐Resistant, Estrogen‐Receptor‐Positive Breast Cancer

Abstract: 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 antihormones are likely to be a composite of the estrogen receptor and growth factor inhibitory activity of these agents. The modulation of growth factor networks during endocrine response is examined, and in vitro and clinical evidence is presented that epidermal growth factor receptor signaling, maintained in either an estrogen receptor‐dependent or a receptor‐independent manner, is critical to antihormone‐resistant breast cancer cell growth. The considerable potential of the epidermal growth factor receptor‐selective tyrosine kinase inhibitor Iressa (ZD 1839) to efficiently treat, and perhaps even prevent, endocrine‐resistant breast cancer is highlighted.

Growth factor signalling networks in breast cancer and resistance to endocrine agents: new therapeutic strategies

Recent evidence demonstrates that growth factor networks are highly interactive with the estrogen receptor (ER) in the control of breast cancer growth and development. As such, tumor responses to anti-hormones are likely to be a composite of the ER and growth factor inhibitory activity of these agents, with alterations/aberrations in growth factor signalling providing a mechanism for the development of anti-hormone resistance. In this light, the current article focuses on illustrating the relationship between growth factor signalling and anti-hormone failure in our in-house tumor models of breast cancer and describes how we are now beginning to successfully target their actions to improve the effects of anti-hormonal drugs and to block aggressive disease progression.

Inhibition of insulin receptor isoform-A signalling restores sensitivity to gefitinib in previously de novo resistant colon cancer cells.

Resistance to antiepidermal growth factor (EGFR) strategies is an emerging clinical problem. Using human colorectal cancer (CRC) cells, we evaluated the involvement of the insulin receptor isoform-A (InsR-A) in de novo resistance to gefitinib, an EGFR tyrosine kinase inhibitor. Challenging the EGFR positive LoVo cells with gefitinib (1 μM) resulted in a small (∼18%) inhibition of cell growth and although a modest reduction in phospho (p)EGFR Tyr845 was seen, pEGFR at residues -Tyr1068 and -Tyr1173 were unchanged. LoVo cells produced unprocessed pro-IGF-1R protein, substantial levels of IGF-II mRNA and mature InsR protein, consisting mainly of the InsR-A isoform. Insulin and IGF-II promoted cell growth and pEGFR Tyr845, Tyr1068 and Tyr1173 activity and conversely, the insulin-like growth factor-1 receptor (IGF-1R)/InsR inhibitor ABDP (1 μM) inhibited growth and reduced pEGFR activity at all three tyrosine residues. pInsR and pAkt levels were increased after gefitinib treatment. Blocking of pInsR with ABDP enabled gefitinib to markedly reduce pEGFR Tyr845, Tyr1068 and Tyr1173. Short-term gefitinib/ABDP dual treatment was more effective than either agent alone and chronic exposure to this combination resulted in total cell loss after 9 weeks, preventing acquisition of resistance to ABDP. LoVo cells with acquired resistance to ABDP were acutely sensitive to gefitinib. We concluded that InsR-A reduces sensitivity to gefitinib in LoVo CRC cells, thus its co-targeting alongside EGFR can improve the anti-tumour effect of gefitinib.

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

IntroductionUpregulation 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.MethodsIn 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.ResultsRAD001 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.ConclusionsThis 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.

Fulvestrant-induced expression of ErbB3 and ErbB4 receptors sensitizes oestrogen receptor-positive breast cancer cells to heregulin β1

IntroductionWe 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.MethodsMCF-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 β1 (HRGβ1) 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.ResultsFulvestrant 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 HRGβ1 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 HRGβ1 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 HRGβ1 in all four cell lines.ConclusionsThese 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.

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

IntroductionRecently 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.MethodsImmunoprecipitation 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 β1 (HRGβ1). 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 HRGβ1 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.ResultsImmunoprecipitation and Western blot analysis revealed an interaction between erbB3 and IRS-1 in MCF-7, T47D and BT-474 cells, with HRGβ1 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 HRGβ1 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 HRGβ1, allowing HRGβ1 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.ConclusionsIRS-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.