Role of endoplasmic reticulum stress induction by the plant toxin, persin, in overcoming resistance to the apoptotic effects of tamoxifen in human breast cancer cells.

BACKGROUND: Persin is a plant toxin that displays synergistic cytotoxicity with tamoxifen in human breast cancer cell lines. Here, we examined the ability of persin to circumvent tamoxifen resistance and delineated the intracellular signalling pathways involved. METHODS: The induction of apoptosis in tamoxifen-resistant and -sensitive breast cancer cells was measured by flow cytometry following treatment with persin±tamoxifen. Markers of endoplasmic reticulum stress (ERS) were analysed following treatment, and their causal role in mediating persin-induced apoptosis was determined using chemical inhibitors and RNA interference. RESULTS: Cells that were resistant to an apoptotic concentration of tamoxifen maintained an apoptotic response to persin. Persin-induced apoptosis was associated with an increase in markers of ERS, that is, CHOP expression and XBP-1 splicing and was decreased by CHOP siRNA. The CASP-4 inhibitor Z-YVAD-FMK markedly inhibited persin-induced apoptosis in both tamoxifen-sensitive and -resistant cells. CONCLUSION: The cytotoxic effects of persin are CASP-4 dependent and mediated by CHOP-dependent and -independent ERS signalling cascades. Increased ERS signalling contributes to persin-induced reversal of tamoxifen resistance.

A novel g.-1258G>A mutation in a conserved putative regulatory element of PAX9 is associated with autosomal dominant molar hypodontia.

Mutations in the transcription factor PAX9 which plays a critical role in the switching of odontogenic potential from the epithelium to the mesenchyme during tooth development cause autosomal dominant non-syndromic hypodontia primarily affecting molars. Linkage analysis on a family segregating autosomal dominant molar hypodontia with markers flanking and within PAX9 yielded a maximum multipoint LOD score of 3.6. No sequence variants were detected in the coding or 5'- and 3'-untranslated regions (UTRs) of PAX9. However, we identified a novel g.-1258G>A sequence variant in all affected individuals of the family but not in the unaffected family members or in 3088 control chromosomes. This mutation is within a putative 5'-regulatory sequence upstream of PAX9 highly conserved in primates, somewhat conserved in ungulates and carnivores but not conserved in rodents. Bioinformatics analysis of the sequence determined that there was no abolition or creation of a putative binding site for known transcription factors. Based on our previous findings that haploinsufficiency for PAX9 leads to hypodontia, we postulate that the g.-1258G>A variant reduces the expression of PAX9 which underlies the hypodontia phenotype in this family.

Overexpression of TFAP2C in invasive breast cancer correlates with a poorer response to anti-hormone therapy and reduced patient survival.

The AP-2gamma transcription factor encoded by the TFAP2C gene is a member of a family of homologous DNA binding proteins that play essential roles during vertebrate embryogenesis but show a restricted pattern of expression in the adult. Elevated expression of the AP-2alpha and AP-2gamma family members has been associated with a number of neoplasms, particularly breast cancer. Here we present an exploratory immunohistochemical study of an archival primary breast tumour series (n = 75) with parallel clinicopathological data using a new, well-characterized antibody to AP-2gamma. Heterogeneous, exclusively nuclear expression of AP-2gamma was found in the epithelial and myoepithelial compartments of normal breast and within tumour epithelial cells. In the breast cancer series, the most notable association was a correlation between elevated levels of AP-2gamma and shortened patient survival (p = 0.0009*). This relationship was also conserved in ER-positive and ErbB2-negative patients; sub-groups generally considered to have a relatively good prognosis. When patient data for survival and duration of treatment response on anti-hormone therapy were examined by multivariate analysis, AP-2gamma was revealed in this study to be an independent predictor of outcome for both survival (p = 0.005) and response to anti-hormone therapy (p = 0.046). Studies using in vitro models confirmed that while tamoxifen response is associated with lower levels of AP-2gamma, acquisition of resistance to this and other anti-hormone measures (eg faslodex or oestrogen deprivation) is associated with high levels of nuclear AP-2gamma. Together these data suggest that elevated tumour AP-2gamma expression can contribute to the failure of cells to growth arrest following anti-hormone treatment and lead to sustained growth and poorer patient outcome.

Development of a food frequency questionnaire for the assessment of quercetin and naringenin intake.

OBJECTIVES: To measure the relationship between quercetin and naringenin intakes as estimated by food frequency questionnaire (FFQ), and the urinary excretion of quercetin and naringenin aglycones after their enzymatic hydrolysis in human volunteers. SUBJECTS AND METHODS: Volunteers were recruited via the Human Nutrition Unit volunteer databank at the Institute of Food Research, Norwich. Sixty-three volunteers were recruited into the study, of which 14 were excluded and 49 completed the study. A modified FFQ was developed and used to estimate daily intake of quercetin and naringenin in 49 healthy volunteers who also provided five 24-h urine samples over a 2-week period. Urinary excretion of quercetin and naringenin metabolites was determined by solid-phase extraction and high-pressure liquid chromatography. RESULTS: The estimated mean intakes of quercetin and naringenin were 29.4 mg (s.d. 15.0) and 58.1 mg (s.d. 62.7) per day, respectively. Mean urinary excretion of quercetin was 60.1 microg (s.d. 33.1) and that of naringenin was 0.56 mg (s.d. 0.4). The correlation between FFQ estimated intake of quercetin and naringenin and levels excreted in the urine were r=0.82 (P<0.0001) and r=0.25 (P=0.05), respectively. CONCLUSIONS: We observed a statistically significant correlation between the urinary excretion of quercetin and naringenin metabolites and their dietary intake as estimated by FFQ. Use of FFQs in epidemiological studies requiring an estimate of flavonoid intake seems justified.

Protein kinase C isoform expression as a predictor of disease outcome on endocrine therapy in breast cancer.

BACKGROUND: Although in vitro breast cancer models have demonstrated a role for protein kinase C (PKC) alpha and delta isoforms in endocrine insensitivity and resistance respectively, there is currently little clinical evidence to support these observations. AIMS: To define the pattern of PKC alpha and delta expression using breast cancer cell lines, with and without endocrine resistance, and also breast cancer samples, where expression can be correlated with clinicopathological and endocrine therapy outcome data. METHODS: PKC isoform expression was examined in tamoxifen responsive, oestrogen receptor positive (ER(+)), ER(+) acquired tamoxifen resistant (TAM-R) and oestrogen receptor negative (ER(-)) cell lines by western blotting and immunocytochemical analysis. PKC isoform expression was then examined by immunohistochemistry in archival breast cancer specimens from primary breast cancer patients with known clinical outcome in relation to endocrine response and survival on therapy. RESULTS: ER(+) breast cancer cell lines expressed considerable PKC-delta but barely detectable levels of PKC-alpha, whereas ER(-) cell lines expressed PKC-alpha but little PKC-delta. ER(+) acquired TAM-R cell lines expressed substantial levels of both PKC-alpha and delta. In clinical samples, high PKC-delta expression correlated to endocrine responsiveness whereas PKC-alpha expression correlated to ER negativity. PKC-delta was an independent predictor of duration of response to therapy. Patients showing a PKC-delta(+)/PKC-alpha(-) phenotype had a six times longer endocrine response than patients with the PKC-delta(+)/ PKC-alpha(+) phenotype (equating to tamoxifen resistance in vitro). CONCLUSIONS: Levels of PKC-alpha and delta expression appear to be indicative of response to anti-oestrogen therapy and could be useful in predicting a patient's suitability for endocrine therapy.

Growth factor signalling in endocrine and anti-growth factor resistant breast cancer.

Growth factors provide powerful mitogenic and survival signals to breast cancer cells and it is therefore not surprising that they are able to subvert inhibitory responses to anti-hormonal drugs. In this review we discuss several mechanisms by which this may be achieved and expand our observations to encompass recently emerging anti-growth factor treatments. The information presented is underpinned by inhibitor studies that show the targeting of such mechanisms in advance of anti-hormone or anti-growth factor resistance development is able to substantially delay this event, thus pointing the way forward to intelligent combination therapies relevant to the future management of breast cancer.

Effects of fulvestrant 250mg in premenopausal women with oestrogen receptor-positive primary breast cancer.

Fulvestrant (Faslodex) reduces markers of hormone sensitivity and proliferation in postmenopausal women. This Phase II double-blind, randomised, multicentre study compared the effects of a single 250mg intramuscular dose of fulvestrant and placebo 14-21 days prior to surgery of curative intent on the oestrogen receptor (ER), progesterone receptor and Ki67 levels in 66 premenopausal women with ER-positive primary breast cancer. There were no statistically significant differences between fulvestrant and placebo with respect to any of the three markers analysed. The most common adverse events in both groups were nausea, headache and pyrexia. Fulvestrant 250mg had no effects on markers of hormone-sensitivity and proliferation in premenopausal women with primary breast cancer when measured at 14-21 days after injection. These findings suggest that a higher fulvestrant dose may be required in this patient population. Further clinical trials are necessary to evaluate the efficacy of fulvestrant in premenopausal women.

Phosphorylation of ERalpha at serine 118 in primary breast cancer and in tamoxifen-resistant tumours is indicative of a complex role for ERalpha phosphorylation in breast cancer progression.

Oestrogen receptor-alpha (ERalpha) is an important prognostic marker in breast cancer and endocrine therapies are designed to inhibit or prevent ERalpha activity. In vitro studies have indicated that phosphorylation of ERalpha, in particular on serine 118 (S118), can result in activation in a ligand-independent manner, thereby potentially contributing to resistance to endocrine agents, such as tamoxifen and aromatase inhibitors. Here we report the immunohistochemistry (IHC) of S118 phosphorylation in 301 primary breast tumour biopsies. Surprisingly, this analysis shows that S118 phosphorylation is higher in more differentiated tumours, suggesting that phosphorylation at this site is associated with a good prognosis in patients not previously treated with endocrine agents. However, we also report that S118 phosphorylation was elevated in tumour biopsies taken from patients who had relapsed following tamoxifen treatment, when compared to pre-treatment biopsies. Taken together, these data are consistent with the view that S118 phosphorylation is a feature of normal ERalpha function and that increases in levels of phosphorylation at this site may play a key role in the emergence of endocrine resistance in breast cancer.

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 microM) resulted in a small ( approximately 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 muM) 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.

Deciphering antihormone-induced compensatory mechanisms in breast cancer and their therapeutic implications.

Breast cancer inhibition by antihormones is rarely complete, and our studies using responsive models reveal the remarkable flexibility of breast cancer cells in recruiting alternative signalling to limit maximal anti-tumour effects of oestrogen receptor alpha (ER) blockade. The recruited mechanism involves antihormone-induced expression of oestrogen-repressed signalling genes. For example, epidermal growth factor receptor gene (EGFR) is induced by antioestrogens and maintains residual kinase and ER phosphorylation, cell survival genes, and thereby allows incomplete antihormone response and emergence of resistance. Microarrays are revealing the breadth of antihormone-induced genes that may attenuate growth inhibition, including NFkappaB, Bag1, 14-3-3zeta and tyrosine kinases, such as HER2 and Lyn. Three concepts are emerging: first, some genes are induced exclusively by antioestrogens, while others extend to oestrogen deprivation; secondly, some are transiently induced, while others persist into resistance; finally, some confer additional adverse features when tumour cells are in an appropriate context. Among the latter is CD59 whose antioestrogen induction may permit evasion of immune surveillance in vivo. Also, induction of pro-invasive genes (including NFkappaB, RhoE and delta-catenin) may underlie our findings that antioestrogens can markedly stimulate migratory behaviour when tumour intercellular contacts are compromised. Based on our promising studies selectively inhibiting EGFR (gefitinib), NFkappaB (parthenolide) or CD59 (neutralising antibody) together with antioestrogens, we propose that co-targeting strategies could markedly improve anti-tumour activity (notably enhancing cell kill) during the antihormone-responsive phase. Furthermore, subverting those induced signalling genes that are retained into resistance (e.g. EGFR, NFkappaB, HER2) may prove valuable in this state. Alongside future deciphering and targeting of genes underlying antioestrogen-promoted invasiveness, embracing of intelligent combination strategies could significantly extend patient survival.

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

We have previously demonstrated that oestrogen receptor alpha (ERalpha) 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 ERalpha 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) ERalpha 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 ERalpha 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 ERalpha, 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 ERalpha 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 ERalpha signalling pathways. Neutralising antibodies to AR inhibited EGFR/ERK1/2 activity, reduced S118 ERalpha phosphorylation and reduced AR mRNA expression in TAM-R cells. These findings suggest that ERalpha 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 ERalpha-mediated production of AR.

Consensus statement. Workshop on therapeutic resistance in breast cancer: impact of growth factor signalling pathways and implications for future treatment.

Anti-hormones (notably tamoxifen), chemotherapy and modern radiotherapeutic approaches are invaluable in the management of breast cancer, and collectively have contributed substantially to the improved survival in this disease. Moreover, there is promise that these successes will continue with the emergence of other endocrine agents (for example, aromatase inhibitors and pure anti-oestrogens). However, de novo and acquired resistance comprises a significant problem with all treatment approaches examined to date. This Workshop aimed to evaluate the contribution made by growth factor signalling pathways in the various resistant states, primarily focusing on resistance to anti-hormonal strategies and spanning experimental models and, where possible, clinical breast cancer data. The successes and limitations of therapeutic targeting of these pathways with various signal transduction inhibitors (STIs) were evaluated in model systems and from emerging clinical trials (including epidermal growth factor receptor inhibitors such as gefitinib). It was concluded that growth factor signalling is an important contributor in the development of endocrine resistance in breast cancer and that use of STIs provides a promising therapeutic strategy for this disease. However, the cancer cell is clearly able to harness alternative growth factor signalling pathways for growth and cell survival in the presence of STI monotherapy and, as a consequence, the efficacy of STIs is likely to be limited by the acquisition of resistance. A number of strategies were proposed from studies in model systems that appeared to enhance anti-tumour actions of existing STI monotherapy, notably including combination therapies targeting multiple pathways. With the increased availability of diverse STIs and improved drug delivery, there is much hope that the more complex therapeutic strategies proposed may ultimately be achievable in clinical practice.

Overview of tyrosine kinase inhibitors in clinical breast cancer.

Studies of cell models and profiling of clinical breast cancer material to reveal the mechanisms of resistance to anti-oestrogen therapy, and to tamoxifen in particular, have reported that this phenomenon can be associated with increased expression and signalling through erbB Type 1 growth factor receptors, notably the epidermal growth factor receptor (EGFR) and HER2. Further molecular studies have revealed an intricate interlinking between such growth factor receptor pathways and oestrogen receptor (ER) signalling. Inhibition of receptor tyrosine kinase activity involved in the EGFR signalling cascade forms the basis for the use of EGFR specific tyrosine kinase inhibitors exemplified by gefitinib (ZD1839, Iressa) and erlotinib (OSI-774, Tarceva). Such agents have proved promising in pre-clinical studies and are currently in clinical trials in breast cancer, where gefitinib has been studied more extensively to date. Here, we present an overview of the current development of gefitinib in clinical breast cancer. This includes results from our clinical breast cancer trial 1839IL/0057 that demonstrate the efficacy of gefitinib within ER-positive, tamoxifen-resistant patients with locally advanced/metastatic disease, where parallel decreases in EGFR signal transduction and the Ki67 (MIB1) proliferation marker can be detected as predicted from model system studies. We also consider trials examining combination treatment with gefitinib and anti-hormonal strategies that will begin to address the clinically important question of whether gefitinib can delay/prevent onset of anti-hormone resistance.

Development of strategies for the use of anti-growth factor treatments.

Aberrant signalling through the epidermal growth factor receptor (EGFR) is associated with increased cancer cell proliferation, reduced apoptosis, invasion and angiogenesis. Over-expression of the EGFR is seen in a variety of tumours and is a rational target for antitumour strategies. Among the classes of agent targeting the EGFR are small-molecule inhibitors, which include gefitinib (IRESSA), which acts by preventing EGFR phosphorylation and downstream signal transduction. De novo and acquired resistance, however, have been reported to gefitinib and here we describe evidence which indicates that the type II receptor tyrosine kinases (RTKs) insulin-like growth factor-I receptor (IGF-IR) and/or insulin receptor (InsR) play important roles in the mediation of responses to gefitinib in the de novo- and acquired-resistance phenotypes in several cancer types. Moreover, combination strategies that additionally target the IGF-IR/InsR can enhance the antitumour effects of gefitinib.

Growth factor signalling and resistance to selective oestrogen receptor modulators and pure anti-oestrogens: the use of anti-growth factor therapies to treat or delay endocrine resistance in breast cancer.

De novo insensitivity and acquired resistance to the selective oestrogen receptor modulator tamoxifen and the pure anti-oestrogen fulvestrant (faslodex) severely limit their effectiveness in breast cancer patients. This is a major clinical problem, since each year upward of 1 million women are dispensed anti-oestrogenic drugs. In order to investigate the phenomenon of anti-oestrogen resistance and to rapidly screen drugs that target the resistance mechanism(s), we have previously established several in vitro breast cancer models that have acquired resistance to anti-hormones. Such cells commonly develop an ability to proliferate after approximately 3 months of exposure to 4-hydroxytamoxifen or fulvestrant, despite an initial endocrine-responsive (i.e. growth-suppressive) phase. The current paper explores the role that growth factor signalling plays in the transition of oestrogen receptor-positive endocrine-responsive breast cancer cells to anti-oestrogen resistance or insensitivity and how we might, in the future, most effectively use anti-growth factor therapies to treat or delay endocrine-resistant states.

Epidermal growth factor receptor/HER2/insulin-like growth factor receptor signalling and oestrogen receptor activity in clinical breast cancer.

Breast cancer models of acquired tamoxifen resistance, oestrogen receptor (ER)+ /ER- de novo resistance and gene transfer studies cumulatively demonstrate the increased importance of growth factor receptor signalling, notably the epidermal growth factor receptor (EGFR)/HER2, in tamoxifen resistance. Our recent in vitro studies also suggest that EGFR signalling productively cross-talks with insulin-like growth factor receptor (IGF-1R) and, where present, activates ER on key AF-1 serine residues to facilitate acquired tamoxifen-resistant growth. This paper presents our immunohistochemical evidence that EGFR/HER2 signalling (i.e. transforming growth factor (TGF)alpha, EGFR and HER2 expression; phosphorylation of EGFR, HER2 and ERK1/2 MAP kinase) is also prominent in clinical de novo resistant and modestly increased in acquired tamoxifen-resistant states, suggesting that anti-EGFR/HER2 strategies may prove valuable treatments. Primary breast cancer samples employed were obtained for (1) patients subsequently treated with tamoxifen for advanced disease where endocrine response and survival data were available and (2) ER+ elderly patients during tamoxifen response and relapse. We also present our clinical immunohistochemical findings that IGF-1R expression, its phosphorylation on tyrosine 1316, and also phosphorylation on serine 118 of ER are not only prominent in ER+ tamoxifen-responsive disease, but are also detectable in ER+ de novo and acquired tamoxifen-resistant breast cancer, where there is evidence of EGFR/ER cross-talk. Our data suggest that agents to deplete effectively ER or IGF-1R signalling may be of value in treating ER+ de novo/acquired tamoxifen resistance in addition to tamoxifen-responsive disease in vivo. IGF-1R inhibitors may also prove valuable in ER- patients, since considerable IGF-1R signalling activity was apparent within approximately 50% of such tumours.

Insulin-like growth factor-I receptor signaling in tamoxifen-resistant breast cancer: a supporting role to the epidermal growth factor receptor.

There is considerable evidence that the epidermal growth factor receptor (EGFR) and IGF-I receptor (IGF-IR) cross-talk in breast cancer cells. In the present study, we have examined whether EGFR/IGF-IR cross-talk exists in EGFR-positive tamoxifen-resistant variants of MCF-7 (Tam-R) and T47D (T47D-R) breast cancer cell lines. Although Tam-R cells expressed reduced IGF-IR protein levels compared with their wild-type MCF-7 counterparts, phosphorylated IGF-IR protein levels were equivalent in the two cell lines under basal growth conditions, possibly as a consequence of increased IGF-II expression in Tam-R cells. IGF-II activated both IGF-IR and EGFR in Tam-R cells, whereas only activation of IGF-IR was observed in wild-type cells. In contrast, epidermal growth factor rapidly induced EGFR, but not IGF-IR, phosphorylation in Tam-R cells. IGF-II promoted direct association of c-SRC with IGF-IR, phosphorylated c-SRC, and increased EGFR phosphorylation at tyrosine 845, a c-SRC-dependent phosphorylation site. Pretreatment with either AG1024 (IGF-IR-specific inhibitor) or an IGF-II neutralizing antibody inhibited basal IGF-IR, c-SRC, and EGFR phosphorylation, and AG1024 significantly reduced Tam-R basal cell growth. The c-SRC inhibitor SU6656 also inhibited growth, reduced basal and IGF-II-induced c-SRC and EGFR phosphorylation, and blocked EGFR activation by TGFalpha. Similarly, in T47D-R cells, AG1024 and SU6656 inhibited basal and IGF-II-induced phosphorylation of c-SRC and EGFR, and SU6656 reduced TGFalpha-induced EGFR activity. These results suggest the existence of a unidirectional IGF-IR/EGFR cross-talk mechanism whereby IGF-II, acting through the IGF-IR, regulates basal and ligand-activated EGFR signaling and cell proliferation in a c-SRC-dependent manner in Tam-R cells. This cross-talk between IGF-IR and EGFR is not unique to Tam-R cells because this mechanism is also active in a tamoxifen-resistant T47D-R cell line.

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.

Growth factor-driven mechanisms associated with resistance to estrogen deprivation in breast cancer: new opportunities for therapy.

There is an increasing body of evidence demonstrating that elevated growth signaling in breast cancer cells can promote forms of endocrine resistance in either an estrogen receptor-dependent or -independent manner. The current article reviews what is known about such growth factor signaling networks and resistance to estrogen withdrawal and considers the many novel therapeutic opportunities that stem from this knowledge.

Insulin-like growth factor-I receptor signalling and acquired resistance to gefitinib (ZD1839; Iressa) in human breast and prostate cancer cells.

De novo and acquired resistance to the anti-tumour drug gefitinib (ZD1839; Iressa), a specific epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) has been reported. We have determined whether signalling through the IGF-I receptor (IGF-1R) pathway plays a role in the gefitinib-acquired resistance phenotype. Continuous exposure of EGFR-positive MCF-7-derived tamoxifen resistant breast cancer cells (TAM-R) to 1 microM gefitinib resulted in a sustained growth inhibition (90%) for 4 months before the surviving cells resumed proliferation. A stable gefitinib-resistant subline (TAM/TKI-R) was established after a further 2 months and this showed no detectable basal phosphorylated EGFR activity. Compared with the parental TAM-R cells, the TAM/ TKI-R cells demonstrated (a) elevated levels of activated IGF-1R, AKT and protein kinase C (PKC)delta, (b) an increased sensitivity to growth inhibition by the IGF-1R TKI AG1024 and (c) an increased migratory capacity that was reduced by AG1024 treatment. Similarly, the EGFR-positive androgen-independent human prostate cancer cell line DU145 was also continuously challenged with 1 microM gefitinib and, although substantial growth inhibition (60%) was seen initially, a gefitinib-resistant variant (DU145/TKI-R) developed after 3 months. Like their breast cancer counterparts, the DU145/TKI-R cells showed increases in the levels of components of the IGF-1R signalling pathway and an elevated sensitivity to growth inhibition by AG1024 compared with the parent DU145 cell line. Additionally, DU145/TKI-R cell migration was also decreased by this inhibitor. We have therefore concluded that in breast and prostate cancer cells acquired resistance to gefitinib is associated with increased signalling via the IGF-1R pathway, which also plays a role in the invasive capacity of the gefitinib-resistant phenotype.