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Zinc has been known to be essential for cell division for over 40 years but the molecular pathways involved remain elusive. Cellular zinc import across biological membranes necessitates the help of zinc transporters such as the SLC39A family of ZIP transporters. We have discovered a molecular process that explains why zinc is required for cell division, involving two highly regulated zinc transporters, as a heteromer of ZIP6 and ZIP10, providing the means of cellular zinc entry at a specific time of the cell cycle that initiates a pathway resulting in the onset of mitosis. Crucially, when the zinc influx across this heteromer is blocked by ZIP6 or ZIP10 specific antibodies, there is no evidence of mitosis, confirming the requirement for zinc influx as a trigger of mitosis. The zinc that influxes into cells to trigger mitosis additionally changes the phosphorylation state of STAT3 converting it from a transcription factor to a protein that complexes with this heteromer and pS38Stathmin, the form allowing microtubule rearrangement as required in mitosis. This discovery now explains the specific cellular role of ZIP6 and ZIP10 and how they have special importance in the mitosis process compared to other ZIP transporter family members. This finding offers new therapeutic opportunities for inhibition of cell division in the many proliferative diseases that exist, such as cancer. Electronic supplementary material The online version of this article (10.1007/s00018-020-03616-6) contains supplementary material, which is available to authorized users.

BackgroundIntratumoural heterogeneity is a poor prognostic feature in triple-negative breast cancer (TNBC) and other high-grade malignancies. It is caused by genomic instability and phenotypic plasticity, but how these features co-evolve during tumour development remains unclear. SOX10 is a transcription factor, neural crest stem cell (NCSC) specifier and candidate mediator of cancer-associated phenotypic plasticity.MethodsUsing immunophenotyping, we investigated the expression of SOX10 in normal human breast tissue and breast cancer (n=21 cosmetic breast reduction and 1,860 tumour samples with clinical annotation). We then defined the context and evolution of its expression in TNBC compared to 21 other malignancies using systems-level transcriptomics.ResultsSOX10 was detected in nuclei of normal mammary luminal progenitor cells, the histogenic origin of most TNBCs. In breast cancer, nuclear SOX10 predicted poor outcome amongst cross-sectional (log-rank p=0.0015, hazard ratio 2.02, n=224) and metaplastic (log-rank p=0.04, n=66) TNBCs. Systems-level transcriptional network analysis identified a core module in SOX10’s normal mammary epithelial transcription program that is rewired to NCSC genes in TNBC. Reprogramming was proportional to DNA damage and genome-wide promoter hypomethylation, particularly at CpG island shores. Using a novel network analysis pipeline, we found that NCSC-like transcriptional reprogramming is also strongly associated with promoter hypomethylation in other SOX10+ malignancies: glioma and melanoma.ConclusionsWe propose that cancer-associated genome hypomethylation simulates the open chromatin landscape of more primitive cell states, and that on this relatively unrestricted background, SOX10 recreates its ancestral gene regulatory circuits by default. These findings provide new insights about the basis of intratumoural heterogeneity and resurrection of developmental phenotypes in cancer; and highlight the potential for therapeutics that limit chromatin remodelling.

Background: It remains clinically important to identify ER positive breast cancers likely to respond to tamoxifen (TAM) and so we aimed to select a group of biomarkers able to predict response. We also assessed whether data from different sample types [tumor microarrays (TMAs) and core biopsies] or tumor sites could be combined for biomarker studies. Methods: A total of 123 endocrine treatment naïve patients with known ER and HER2 status treated with TAM had paraffin-embedded tumor tissue available either as TMAs (n=102) or core biopsies (n=21). TMA cores were collected from three different tumor sites, two central and one peripheral. Ten biomarkers were evaluated by immunohistochemistry, for % positivity and/or H-Score, comprising: ER, HER2, Ki67, phosphorylated forms of ER (Ser118), IGF1R, PRAS40, Akt & MAPK (ERK1/2), and PTEN & androgen receptor expression (AR). Each tumor was analysed for Akt1 E17K somatic mutation using BEAMing technology. Patient outcome was assessed by clinical benefit (CB) rate & survival analyses [time to progression (TTP) and time to death (TTD)]. Results: There was no significant difference in % positivity or H-Score between central & peripheral tumor sites for all biomarkers examined. After False Discovery Rate (FDR) correction differences (P

Purpose: The STAKT study examined short-term exposure (4.5 days) to the oral selective pan-AKT inhibitor capivasertib (AZD5363) to determine if this drug can reach its therapeutic target in sufficient concentration to significantly modulate key biomarkers of the AKT pathway and tumor proliferation. Patients and Methods: STAKT was a two-stage, double-blind, randomized, placebo-controlled, “window-of-opportunity” study in patients with newly diagnosed ER+ invasive breast cancer. Stage 1 assessed capivasertib 480 mg b.i.d. (recommended monotherapy dose) and placebo, and stage 2 assessed capivasertib 360 and 240 mg b.i.d. Primary endpoints were changes from baseline in AKT pathway markers pPRAS40, pGSK3β, and proliferation protein Ki67. Pharmacologic and pharmacodynamic properties were analyzed from blood sampling, and tolerability by adverse-event monitoring. Results: After 4.5 days' exposure, capivasertib 480 mg b.i.d. (n = 17) produced significant decreases from baseline versus placebo (n = 11) in pGSK3β (H-score absolute change: −55.3, P = 0.006) and pPRAS40 (−83.8, P < 0.0001), and a decrease in Ki67 (absolute change in percentage positive nuclei: −9.6%, P = 0.031). Significant changes also occurred in secondary signaling biomarker pS6 (−42.3, P = 0.004), while pAKT (and nuclear FOXO3a) also increased in accordance with capivasertib's mechanism (pAKT: 81.3, P = 0.005). At doses of 360 mg b.i.d. (n = 5) and 240 mg b.i.d. (n = 6), changes in primary and secondary biomarkers were also observed, albeit of smaller magnitude. Biomarker modulation was dose and concentration dependent, and no new safety signals were evident. Conclusions: Capivasertib 480 mg b.i.d. rapidly modulates key biomarkers of the AKT pathway and decreases proliferation marker Ki67, suggesting future potential as an effective therapy in AKT-dependent breast cancers.

Endocrine therapy resistance frequently develops in estrogen receptor positive (ER+) breast cancer, but the underlying molecular mechanisms are largely unknown. Here, we show that 3-dimensional (3D) chromatin interactions both within and between topologically associating domains (TADs) frequently change in ER+ endocrine-resistant breast cancer cells and that the differential interactions are enriched for resistance-associated genetic variants at CTCF-bound anchors. Ectopic chromatin interactions are preferentially enriched at active enhancers and promoters and ER binding sites, and are associated with altered expression of ER-regulated genes, consistent with dynamic remodelling of ER pathways accompanying the development of endocrine resistance. We observe that loss of 3D chromatin interactions often occurs coincidently with hypermethylation and loss of ER binding. Alterations in active A and inactive B chromosomal compartments are also associated with decreased ER binding and atypical interactions and gene expression. Together, our results suggest that 3D epigenome remodelling is a key mechanism underlying endocrine resistance in ER+ breast cancer., Endocrine therapy resistance occurs often in estrogen receptor positive (ER+) breast cancer. Here, the authors find that 3D epigenome remodelling at ER-bound enhancer-promoter interactions is a key mechanism underlying endocrine resistance.

Background\ud \ud MicroRNAs are potent post-transcriptional regulators involved in all hallmarks of cancer. Mir-196a is transcribed from two loci and has been implicated in a wide range of developmental and pathogenic processes, with targets including Hox, Fox, Cdk inhibitors and annexins. Genetic variants and altered expression of MIR196A are associated with risk and progression of multiple cancers including breast cancer, however little is known about the regulation of the genes encoding this miRNA, nor the impact of variants therein.\ud \ud Methods\ud \ud Genomic data and chromatin interaction analysis were used to discover functional promoter and enhancer elements for MIR196A. Expression data were used to associate MIR196A with mechanisms of resistance, breast cancer subtypes and prognosis.\ud \ud Results\ud \ud Here we demonstrate that MIR196A displays complex and dynamic expression patterns, in part controlled by long-range transcriptional regulation between promoter and enhancer elements bound by ERα. Expression of this miRNA is significantly increased in drug-resistant models of hormone-receptor positive disease. The expression of MIR196A also proves to be a robust prognostic factor for patients with advanced and post-menopausal ER+ disease.\ud \ud Conclusion\ud \ud This work sheds light on the normal and abnormal regulation of MIR196A and provides a novel stratification method for therapeutically resistant breast cancer.

Despite the effectiveness of endocrine therapies to treat estrogen receptor-positive (ER+) breast tumours, two thirds of patients will eventually relapse due tode novoor acquired resistance to these agents. Cancer Stem-like Cells (CSCs), a rare cell population within the tumour, accumulate after anti-estrogen treatments and are likely to contribute to their failure. Here we studied the role of p21-activated kinase 4 (PAK4) as a promising target to overcome endocrine resistance and disease progression in ER+ breast cancers. PAK4 predicts for resistance to tamoxifen and poor prognosis in 2 independent cohorts of ER+ tumours. We observed that PAK4 strongly correlates with CSC activity in metastatic patient-derived samples irrespective of breast cancer subtype. However, PAK4-driven mammosphere-forming CSC activity increases alongside progression only in ER+ metastatic samples. PAK4 activity increases in ER+ models during acquired resistance to endocrine therapies. Targeting PAK4 with either CRT PAKi, a small molecule inhibitor of PAK4, or with specific siRNAs abrogates CSC activity/self-renewal in clinical samples and endocrine-resistant cells. Together, our findings establish that PAK4 regulates stemness during disease progression and that its inhibition reverses endocrine resistance in ER+ breast cancers.HighlightsPAK4 predicts for failure of endocrine therapies and poor prognosisPAK4 drives stemness and progression in ER+ metastatic breast cancerTargeting PAK4 abrogates breast CSC activity and restores sensitivity to endocrine treatmentsTargeting PAK4 will improve outcome of ER+ breast cancer patientsList of Abbreviations that appeared in abstractCancer Stem-like Cells (CSCs)p21-activated kinase 4 (PAK4)Estrogen Receptor (ER)

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.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.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.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.

Purpose: One third of ER-positive breast cancer patients who initially respond to endocrine therapy become resistant to treatment. Such treatment failure is associated with poor prognosis and remains an area of unmet clinical need. Here, we identify a specific posttranslational modification that occurs during endocrine resistance and which results in tumor susceptibility to the apoptosis-inducer TRAIL. This potentially offers a novel stratified approach to targeting endocrine-resistant breast cancer. Experimental Design: Cell line and primary-derived xenograft models of endocrine resistance were investigated for susceptibility to TRAIL. Tumor viability, cancer stem cell (CSC) viability (tumorspheres), tumor growth kinetics, and metastatic burden were assessed. Western blots for the TRAIL-pathway inhibitor, c-FLIP, and upstream regulators were performed. Results were confirmed in primary culture of 26 endocrine-resistant and endocrine-naïve breast tumors. Results: Breast cancer cell lines with acquired resistance to tamoxifen (TAMR) or faslodex were more sensitive to TRAIL than their endocrine-sensitive controls. Moreover, TRAIL eliminated CSC-like activity in TAMR cells, resulting in prolonged remission of xenografts in vivo. In primary culture, TRAIL significantly depleted CSCs in 85% endocrine-resistant, compared with 8% endocrine-naïve, tumors, whereas systemic administration of TRAIL in endocrine-resistant patient-derived xenografts reduced tumor growth, CSC-like activity, and metastases. Acquired TRAIL sensitivity correlated with a reduction in intracellular levels of c-FLIP, and an increase in Jnk-mediated phosphorylation of E3-ligase, ITCH, which degrades c-FLIP. Conclusions: These results identify a novel mechanism of acquired vulnerability to an extrinsic cell death stimulus, in endocrine-resistant breast cancers, which has both therapeutic and prognostic potential. Clin Cancer Res; 24(10); 2452–63. ©2018 AACR.

Background: The annual recurrence rates of post-menopausal ER-positive breast cancers persist beyond the first 5 years of diagnosis and treatment and the mortality rates in this period are higher versus ER-negative cancers. Extended endocrine therapy past 5 years has been shown to be of benefit but is associated with increased toxicity and cost thus the ability to predict patients who are at highest risk of late relapse would be of significant benefit in clinical decision making in this context. β-catenin is an intracellular protein that undergoes Wnt-mediated nuclear translocation where it transactivates genes implicated in tumour development and progression. We have also previously reported that β-catenin can play a role in aggressive resistance that accompanies prolonged endocrine treatment in vitro. In this study, we thus investigated whether β-catenin expression in ER+ breast cancer was predictive of recurrence beyond 5 years in an analysis of three separate endocrine-treated cohorts. Methods: Associations between β-catenin gene expression and relapse free survival (RFS) were performed using the online KMplotter tool. Immunostaining for total β-catenin was performed on tissue samples from 3 ER+ primary breast cancer series with long-term follow-up data: Nottingham 2000 (n=384, tamoxifen-only); ABC (n=570; tamoxifen only); transATAC (n=743; tamoxifen or ansatrozole). The association between subcellular (nuclear or cytoplasmic) β-catenin expression and RFS was determined in (i) the entire cohort, (ii) the first 5 years of tamoxifen treatment versus post-5 years. Results: KMplotter analysis of β-catenin in ER+, tamoxifen-treated patient samples (n=665) revealed a significant relationship with improved RFS in the post-five year cohort [HR: 0.48 (0.34-0.68); p=0.000019) versus the first 5 years [HR: 0.91 (0.61-1.36; p=0.64)]. No significant association was observed in untreated ER+ patients. In the Nottingham series, nuclear β-catenin positivity was significantly associated with improved survival in the 20-year follow-up for tamoxifen-treated patients (p=0.047) but not in the first five years (p=0.239). Cytoplasmic β-catenin did not associate with survival. Further analysis in the ABC series revealed an association between nuclear β-catenin positivity and improved survival for tamoxifen-treated patients in the entire cohort (HR: 0.52 (0.18, 0.55); p=0.00005). However, nuclear β-catenin was more strongly associated with improved outcome in the post 5-year treatment group (HR: 0.30 (0.14, 0.66); p=0.01) versus the first five years of treatment (HR: 0.33 (0.15,0.73); p=0.06). No significant associations were seen with cytoplasmic β-catenin. The transATAC trial material again revealed an association between presence of nuclear β-catenin and reduced distant recurrence in the post 5-years endocrine-treated cohort (HR: 0.54 (0.33, 0.91): x2=5.52, p=0.018) versus years 1-5 (HR: 1.16 (0.67, 2.01); x2=0.29, p=0.59). Conclusions: This is the first study to demonstrate that nuclear β-catenin may represent a predictive biomarker for late relapse following tamoxifen treatment in ER+ breast cancer where, contrary to traditional hypotheses and pre-clinical data, its nuclear expression is strongly associated with good outcome post-five years of treatment. Citation Format: Stephen Hiscox, Chris Smith, Robert I Nicholson, Julia Gee, Adrian Harris, Judith Bliss, Eleftheria Kalaizak, Ivana Sestak, Mitch Dowsett, Jack Cuzick, Ian Ellis, Peter Barrett-Lee. Nuclear β-catenin negativity predicts for late relapse in ER+, tamoxifen-treated breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P3-06-01.

Background: Oestrogen receptor positive (ER+) breast cancer patients can acquire endocrine resistance and 10-20% tumours have lost ER at relapse. While growth factor pathway hyperactivity and ER promoter methylation contribute to de novo ER negativity, ER loss in acquired resistance is largely unexplored. We have recently developed 11 lines from MCF7, T47D, BT474 & MDAMB361 cells to model acquired resistance emerging with prolonged (3 year) endocrine treatment both in ER+/HER2- and ER+/HER2+ disease. Here we establish prevalence of acquired ER loss in the panel, determine any association with aggressiveness, and explore ER loss mechanisms in acquired endocrine resistance. Methods: Authenticated acquired resistant models derived from endocrine responsive lines cultured for 3 years with 10-7M tamoxifen (TamR), 10-7M fulvestrant (FasR) or oestrogen deprivation (5% charcoal-stripped foetal calf serum SFCSR) were profiled for ER & PR by PCR, immunocytochemistry and Western blotting (+/- 1-2wk antihormone withdrawal), for 2nd-line endocrine responsiveness and for migration using Boyden chamber assays vs. time-matched controls. Src kinase, EGFR, HER2, MAPK & AKT activity were examined and whether their respective inhibition using saracatinib or gefitinib (1μM), trastuzumab (100nM), U0126 or LY294002 (5μM) for 1wk restored ER. ESR1 promoter methylation was examined by bisulfite modification & MethyLight PCR. Results: Substantial ER mRNA & protein loss occurred in 7/11 long-term acquired endocrine resistant lines. This was irreversible by antihormone withdrawal and paralleled by complete PR loss and endocrine growth-insensitivity. While seen in all fulvestrant resistant lines, ER loss was less frequent with tamoxifen (in MCF7TamR & MDATamR) and only seen in oestrogen deprived resistant T47DSFCSR cells. Increased migration accompanied acquired ER loss in ER+/HER2- derived MCF7TamR, MCF7FasR & T47DSFCSR cells and was saracatinib-sensitive. Src and further growth factor pathway activity increased in several acquired resistant models, and ER loss associated with increased EGFR/HER2 in the MCF7- & MDA-derived cells and with increased MAPK activity in all lines. Weak ER recovery was seen in antioestrogen resistant models treated with saracatinib (MDATamR, MCF7FasR), gefitinib (MDATamR, BT474FasR, MCF7FasR/TamR) or trastuzumab (MCF7TamR). ESR1 DNA methylation was only prominent in MDATamR and MCF7TamR cells. No inhibitor restored ER in the T47D-derived cells, including T47DSFCSR which also lacked ESR1 methylation. Conclusions: Although ER loss is very prominent in this acquired resistant cell panel, it demonstrates there is capacity of prolonged antihormones, chiefly antioestrogens, to promote receptor loss independent of initial HER2 status. Acquired ER loss clinically would be expected to confer endocrine insensitivity and poorer prognosis given the panel findings. Where ER loss emerged with antioestrogens there was some mechanistic-overlap with de novo ER negativity, including ER promoter methylation for acquired tamoxifen resistance. Our future studies will use the panel to address if targeting these mechanisms can be optimized for ER recovery or if further mechanisms also drive ER loss in acquired resistance, notably for prolonged oestrogen deprivation. Citation Format: Julia M Gee, Mei Yee Meng, Richard A McClelland, Huw J Mottram, Susan R Kyme, Pauline Finlay, Lindy Goddard, Carol M Dutkowski, Denise Barrow, Walther Parson, Heidi Fiegl. A new cell panel to study oestrogen receptor loss in acquired endocrine resistant breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P3-05-19.

Predicting response to endocrine therapy and survival in oestrogen receptor positive breast cancer is a significant clinical challenge and novel prognostic biomarkers are needed. Long-range regulators of gene expression are emerging as promising biomarkers and therapeutic targets for human diseases, so we have explored the potential of distal enhancer elements of non-coding RNAs in the prognostication of breast cancer survival. HOTAIR is a long non-coding RNA that is overexpressed, promotes metastasis and is predictive of decreased survival. Here, we describe a long-range transcriptional enhancer of the HOTAIR gene that binds several hormone receptors and associated transcription factors, interacts with the HOTAIR promoter and augments transcription. This enhancer is dependent on Forkhead-Box transcription factors and functionally interacts with a novel alternate HOTAIR promoter. HOTAIR expression is negatively regulated by oestrogen, positively regulated by FOXA1 and FOXM1, and is inversely correlated with oestrogen receptor and directly correlated with FOXM1 in breast tumours. The combination of HOTAIR and FOXM1 enables greater discrimination of endocrine therapy responders and non-responders in patients with oestrogen receptor positive breast cancer. Consistent with this, HOTAIR expression is increased in cell-line models of endocrine resistance. Analysis of breast cancer gene expression data indicates that HOTAIR is co-expressed with FOXA1 and FOXM1 in HER2-enriched tumours, and these factors enhance the prognostic power of HOTAIR in aggressive HER2+ breast tumours. Our study elucidates the transcriptional regulation of HOTAIR, identifies HOTAIR and its regulators as novel biomarkers of patient response to endocrine therapy and corroborates the importance of transcriptional enhancers in cancer.

While endocrine therapy is the mainstay of ER+ breast cancer, the clinical effectiveness of these agents is limited by the phenomenon of acquired resistance that is associated with disease relapse and poor prognosis. Our previous studies revealed that acquired resistance is accompanied by a gain in cellular invasion and migration and also that CD44 family proteins are overexpressed in the resistant phenotype. Given the association of CD44 with tumor progression, we hypothesized that its overexpression may act to promote the aggressive behavior of endocrine-resistant breast cancers. Here, we have investigated further the role of two specific CD44 isoforms, CD44v3 and CD44v6, in the endocrine-resistant phenotype. Our data revealed that overexpression of CD44v6, but not CD44v3, in endocrine-sensitive MCF-7 cells resulted in a gain in EGFR signaling, enhanced their endogenous invasive capacity, and attenuated their response to endocrine treatment. Suppression of CD44v6 in endocrine-resistant cell models was associated with a reduction in their invasive capacity. Our data suggest that upregulation of CD44v6 in acquired resistant breast cancer may contribute to a gain in the aggressive phenotype of these cells and loss of endocrine response through transactivation of the EGFR pathway. Future therapeutic targeting of CD44v6 may prove to be an effective strategy alongside EGFR-targeted agents in delaying/preventing acquired resistance in breast cancer.

Cyclin E2, but not cyclin E1, is included in several gene signatures that predict disease progression in either tamoxifen-resistant or metastatic breast cancer. We therefore examined the role of cyclin E2 in antiestrogen resistance in vitro and its potential for therapeutic targeting through cyclin-dependent kinase (CDK) inhibition. High expression of CCNE2, but not CCNE1, was characteristic of the luminal B and HER2 subtypes of breast cancer and was strongly predictive of shorter distant metastasis-free survival following endocrine therapy. After antiestrogen treatment of MCF-7 breast cancer cells, cyclin E2 mRNA and protein were downregulated and cyclin E2–CDK2 activity decreased. However, this regulation was lost in tamoxifen-resistant (MCF-7 TAMR) cells, which overexpressed cyclin E2. Expression of either cyclin E1 or E2 in T-47D breast cancer cells conferred acute antiestrogen resistance, suggesting that cyclin E overexpression contributes to the antiestrogen resistance of tamoxifen-resistant cells. Ectopic expression of cyclin E1 or E2 also reduced sensitivity to CDK4, but not CDK2, inhibition. Proliferation of tamoxifen-resistant cells was inhibited by RNAi-mediated knockdown of cyclin E1, cyclin E2, or CDK2. Furthermore, CDK2 inhibition of E-cyclin overexpressing cells and tamoxifen-resistant cells restored sensitivity to tamoxifen or CDK4 inhibition. Cyclin E2 overexpression is therefore a potential mechanism of resistance to both endocrine therapy and CDK4 inhibition. CDK2 inhibitors hold promise as a component of combination therapies in endocrine-resistant disease as they effectively inhibit cyclin E1 and E2 overexpressing cells and enhance the efficacy of other therapeutics. Mol Cancer Ther; 11(7); 1488–99. ©2012 AACR.

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.

Background: Various molecular markers assessed by IHC (ER, PR, HER2) and gene expression profiling (e.g. Oncotype Dx) have been developed as prognostic and predictive tools for breast cancer. Gene profiling is said to be superior to IHC but at a considerable cost with limited availability. IHC is relatively inexpensive and more readily available. If early breast cancer patients who are going to relapse within 5 years of curative surgery despite adjuvant chemotherapy could be identified by IHC on FFPE tissue alternative adjuvant therapies could be explored. In this context, here we evaluate IHC for expression of a panel of molecular markers implicated in: growth signalling pathways (ER, PR, HER2, EGFR, CD71, Ki67, MCM2), cell survival (Bcl-2, Bag 1), angiogenesis (PDGFRa) and cell cycle progression (Aurora A, MCM2). Of note, this study includes markers of breast cancer molecular subtype (ER, PR, HER2, Ki67, EGFR, also CK5/6) and several proteins encoded by genes in the Oncotype Dx test (ER, PR, HER2, Ki 67, Bcl2, Bag1 and CD68). Materials and Method: 72 cases (R) relapsing within 5 years of curative surgery, 72 controls (C), relapse free > 5 years were identified from the hospital records. All patients had adjuvant chemotherapy. Controls were matched to cases by Adjuvant! recurrence risk (ARR). Optimised IHC was performed on FFPE TMA slides using a Ventana autostainer. Protein expression was evaluated on digitalised images (Mirax scanner). Survival analysis by molecular markers expression and also 5 molecular subtypes, Luminal A (LA = ER/PR+, HER2−, Ki67-), Luminal B (LB = ER/PR+, HER2/Ki67+), HER2 enriched (H = ER-, PR-, HER2+), Core Basal (CB = ER-, PR-, HER2−, CK5/6/EGFR+) and 5-negative (5N = negative for ER,PR,HER2,EGFR,CK5/6)], were performed. SPSS 16v. was used for statistical analysis. Findings: All but four cases had died at the time of analysis. Four controls developed relapse at 83.8, 90.6, 107.7, 127.6 months respectively. Two controls died from non-breast cancer causes. Median (m) follow-up for the controls group (ie. mOS)was 104.9 mo (72.8 - 164.4). For cases, mRFS and mOS were 23.2 (4.5 - 59.9) and 39.7(8.1 - 129). mRFS and mOS for IHC molecular subtypes were: Subtype LA = not yet & not yet; LB = 58.1 & 86.1; CB = 15.4 & 30.4; H = 28 & 55.9; 5N = 19.9 & 26 (p < 0.0001 & Discussion: Subtypes CB & 5N, negative Bcl-2 & MCM2, positive Aurora A & PDGFRa expression as measured by IHC were predictive of poor RFS and OS. While these findings need to be verified in an independent cohort, IHC profiles nevertheless have potential to stratify different risk groups for clinical trials and effective adjuvant treatments. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-07-21.

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.

Background: CD71 is involved in the cellular uptake of iron and is expressed on cells with high proliferation. Interestingly, we have Interestingly, we shown that its expression is associated with elevated tumour proliferation, shortened breast cancer specific survival and worsened outlook in ER+ adjuvant tamoxifen treated early breast cancer patients (Habashy et al, 2010). Here we extend our studies of the relationship between CD71 and tamoxifen outcome by study of an exploratory series of patients where endocrine response was directly assessable. Material and Methods: CD71 (clone 10F11 antibody; Abcam, Cambridge, UK) and proliferation (MIB1) immunohistochemistry were performed as described in Habashy et al (2010) on 87 formalin fixed paraffin-embedded breast cancers. These were obtained from patients within the Nottingham Tenovus Multiple Antibody Study who had received systemic tamoxifen therapy for locally advanced primary carcinoma or metastatic disease or for recurrence after surgery alone. All had lesions assessable for response quality at 6 months, with survival and duration of antihormone response measured from initiation of antihormone to death or progression on therapy respectively. 48 tumours were classified as ER+ and 39 ER-. Results: Within this tamoxifen treated series, 68/87 patients were deemed positive for CD71 (plasma membrane and cytoplasmic), showing HScore values >=5. In the ER+ subgroup, a greater clinical response rate to tamoxifen was seen in CD71 — tumours (10 CR/PR, 3 S and 2 P) versus their CD71+ counterparts (CR/PR, 11 S and 16 P, p=0.015). CD71 positivity was associated with a significantly shortened time to progression and death (Kaplan Meier Test, p=0.026 and p=0.005 respectively). Examination of the proliferation marker MIB1 in ER+ tumours revealed a positive association with CD71 expression (Spearman's Test, p=0.01). In ER-patients, comprising predominantly progressive disease on tamoxifen therapy, CD71 status showed no impact on either time to progression of the disease or death. Discussion: The present study extends our novel findings relating CD71 expression to loss of response to tamoxifen in ER+ breast cancer and reveals CD71 associates with increased tumour cell proliferation in clinical disease. Whether CD71 plays a causative role in the direction of these events and can be targeted to improve endocrine response requires to be ascertained. Habashy HO et al, 2010, Breast Cancer Research and Treatment 119:283-293 Abd El-Rehim DM et al, 2005, Int J Cancer 116: 340-350 Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P2-06-19.

Background: Gene signatures including OncotypeDX are under intense study for their prognostic value, but there is also interest in their predictive value for outcome on endocrine therapy and whether markers reflected by such signatures retain predictive value when measured immunohistochemically (IHC). OncotypeDX comprises 16 cancer (& 5 reference) genes, including oestrogen receptor (ER)-related genes, HER2 pathway and proliferation/cell cycle-associated genes. A further gene in OncotypeDX, bcl-2-associated athanogene 1 (BAG-1), is understudied in breast cancer but increased IHC for BAG-1 has recently been related to improved tamoxifen outcome (Millar et al. Br J Cancer 100, 123-33, 2009). Here we examine if IHC for progesterone receptor (PR), HER2 and the proliferation-associated protein Ki67 can discriminate adjuvant tamoxifen outcome in an ER+ primary breast cancer series. We also evaluate BAG-1 in this series, and in ER+ breast cancer cell lines during antihormone treatment and following acquisition of resistance, to further consolidate relation of BAG-1 IHC to antihormone outcome. Material and Methods: IHC for PR, HER2, Ki67 (DAKO clone MIB1) and BAG-1 (Chemicon MAB4611) was performed in FFPE tissue microarrays from tamoxifen-treated ER+ primary breast cancer patients with 10 year follow-up (n=383). Clinicopathological data, including disease-free interval (DFI) and breast cancer-specific survival (BCSS), were available for statistical analysis. BAG-1 IHC was also performed on paraffin-embedded cell pellets prepared from ER+ breast cancer lines (MCF-7, T47D) after 10 days culture with 10-7M 4OH-tamoxifen (4OHT) and following acquisition of resistance. Results: Kaplan Meier analysis demonstrated that increased NPI, PR negativity, HER2 overexpression and Ki67 positivity (>=10%) could discriminate ER+ tamoxifen-treated patients with reduced DFI (P70%) had significantly poorer outcome (DFI & BCSS: P Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P2-09-37.

ONCOPOOL is a retrospectively compiled database of primary operable invasive breast cancers treated in the 1990s in 10 European breast cancer Units. Sixteen thousand and nine hundred and forty four cases were entered, with tumours less than 5 cm diameter in women aged 70 or less (mean age 55). Data Data were date of birth, mode of diagnosis, pathology (size, lymph node status, grade, type, lympho-vascular invasion and hormone receptor) and therapies and outcome measures: first local, regional or distant recurrences, contralateral primary, date and cause of death. Tumour characteristics Mean diameter 1.8 cm, 66% lymph node negative, 24% 1–3 lymph nodes involved and 10% had 4 or more involved. Grade 1, 29%; Grade 2, 41%; and Grade 3, 30%. Polynomial relationships were established between grade, stage and size. Seventy-five percent were oestrogen receptor (ER) positive. ER closely related to grade. Outcomes Overall Survival was 89% at 5 years from diagnosis, 80% 10 years and 73% 15 years; Breast Cancer-Specific survivals were 91%, 84% and 79%. Survival strongly related to the Nottingham Prognostic Index (NPI).Cases detected at screening had 84% 10-year survival, those presenting symptomatically 76%. ER positive cases treated with adjuvant hormone therapy had a reduction in risk of death of 13% over those not receiving adjuvant therapy (p = 0.000). ER negative cases treated with chemotherapy showed a risk reduction of 23% over those not receiving chemotherapy (p = 0.000).

Estrogen receptor (ER)-positive acquired tamoxifen-resistant (TAM-R) MCF-7 breast cancer cell lines exhibit epidermal growth factor receptor (EGFR) expression/signaling and are growth-inhibited by gefitinib (IRESSA). We examined the effect of gefitinib on ER-positive TAM-R and ER-negative hormone-insensitive breast cancer in a Phase II study. Fifty-four patients with breast cancer [ER-positive/acquired TAM-R (n = 28); ER-negative (n = 26)] received oral gefitinib 500 mg/day. Tumor biopsies were taken pre- (n = 28) and 8 weeks post-treatment (n = 14 matched samples). Gefitinib was well tolerated and the clinical benefit rate (objective response or stable disease >24 weeks) was 33.3% overall (n = 18/54), and 53.6 and 11.5% in ER-positive/TAM-R and ER-negative patients, respectively. Pretreatment ER and progesterone receptor-positivity were associated with response (p 10% decline in EGFR phosphorylation demonstrated parallel decreases in ERK1/2 MAPK phosphorylation. Acquired tamoxifen resistance appears in part mediated through EGFR signaling and can be blocked with gefitinib.

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 10 years 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.

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.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.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.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).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 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.

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 250 mg 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 250 mg 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.

Expression of oestrogen receptor (ESR1) determines whether a breast cancer patient receives endocrine therapy, but does not guarantee patient response. The molecular factors that define endocrine response in ESR1-positive breast cancer patients remain poorly understood. Here we characterize the DNA methylome of endocrine sensitivity and demonstrate the potential impact of differential DNA methylation on endocrine response in breast cancer. We show that DNA hypermethylation occurs predominantly at oestrogen-responsive enhancers and is associated with reduced ESR1 binding and decreased gene expression of key regulators of ESR1 activity, thus providing a novel mechanism by which endocrine response is abated in ESR1-positive breast cancers. Conversely, we delineate that ESR1-responsive enhancer hypomethylation is critical in transition from normal mammary epithelial cells to endocrine-responsive ESR1-positive cancer. Cumulatively, these novel insights highlight the potential of ESR1-responsive enhancer methylation to both predict ESR1-positive disease and stratify ESR1-positive breast cancer patients as responders to endocrine therapy., The molecular factors influencing patient response to endocrine therapy are poorly understood. Here Stone et al. characterize the DNA methylome of endocrine response and show that methylation of oestrogen receptor-associated enhancers underpins endocrine sensitivity in human breast cancer.

Oestrogen receptor-α (ERα) is an important prognostic marker in breast cancer and endocrine therapies are designed to inhibit or prevent ERα activity. In vitro studies have indicated that phosphorylation of ERα, 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 ERα 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.

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.

Over-expression or aberrant signalling of the erbB family members epidermal growth factor receptor (EGFR) and HER2 (erbB2/neu) have been associated with the pathogenesis of the malignant phenotype. In addition, high levels of EGFR and HER2 expression have been shown to correlate with poor prognosis and also implicated in disease progression. Signal transduction inhibitors (STIs) have been developed with specifically target these receptors and include the small molecule tyrosine kinase inhibitor gefitinib (IressaTM) which targets the EGFR and the humanised monoclonal antibody trastuzumab (HerceptinTM), which has anti-tumour activity against HER2. Studies however, have indicated that de novo or acquired resistance to these agents is a major clinical problem. Cancer cells are highly adaptive and can readily switch from one receptor signalling pathway to another in order to maintain growth or cell survival, a process paradoxically, that in many instances is induced by the anti-tumour agents themselves, ultimately limiting their activity and promoting resistance. Evidence is accumulating which demonstrates that signalling interplay occurs between the EGFR/HER2 and the insulin-like growth factor -1 receptor (IGF-1R) and the article will focus on the growth factor pathway switching that occurs between these receptors which can influence the effectiveness gefitinib and trastuzumab.

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 TGFα. Similarly, in T47D-R cells, AG1024 and SU6656 inhibited basal and IGF-II-induced phosphorylation of c-SRC and EGFR, and SU6656 reduced TGFα-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.

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)α, 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 ~50% of such tumours.

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.

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.

Disease-free interval differs amongst invasive breast cancer patients, who had not received any prior adjuvant systemic therapy, with oestrogen receptor (ER) positive cancers treated by endocrine therapy at the time of relapse. This suggests the presence of biological factors inherent in tumours which affect responsiveness to subsequent endocrine therapy. ER splice variants result from exon deletions and can repress wild-type receptors and modulate anti-oestrogen activity. We hypothesise ER splice variants contribute to the differences seen with therapeutic response in these patients. We have characterised variant ER expression in primary invasive breast cancer patients that either responded or not responded to endocrine therapy at the time of relapse. Breast tumour cells were isolated from formalin-fixed archival tumour sections using laser microdissection. Total RNA was extracted and expression of ER�, ER�R2-3, ER�R3, ER�R5, ER�1, ER�2 was quantified using real-time PCR. Gene expression was normalised against 18s expression. Expression of ER� wildtype and variants were detected in most breast tumours although levels differed. ER�1 was highly expressed in tumours with low ER� expression. ER�2 was less commonly expressed. An association between disease-free interval and ER variant expression remains to be elucidated although there was a tendency for tumours expressing ER� also expressed ER�R3.

Background: In patients (pts) with breast cancer, increased epidermal growth factor receptor (EGFR)/HER2 signalling is associated with de novo hormone resistance and oestrogen receptor (ER)-negative phenotypes. EGFR signalling is a key growth regulator in ER-positive acquired tamoxifen-resistant (TAM-R) models where EGFR-TKIs (EGFR tyrosine kinase inhibitors) are potent inhibitory agents. Preliminary clinical data show that the EGFR-TKI gefitinib (‘Iressa’, ZD1839) may be an effective treatment for TAM-R breast cancer (Proc Am Soc Clin Oncol 2003; 22: 7, abs 23). Here we report on expression of ER, EGFR, HER2 and insulin-like growth factor receptor (IGF-1R) in pts enrolled in a Phase II study of gefitinib. Methods: Two groups of pts were recruited: an ER-positive TAM-R group (n=20) and an ER-negative group (n=27). Pts received oral gefitinib 500 mg/day. Immunostaining was performed for ER and EGFR expression and HER2/IGF-1R expression/phosphorylation, and was semiquantified by HScore. With the exception of ER, median HScore was used as the positivity cut-off point. SPSS statistical analysis was used to determine if marker expression predicted gefitinib response by UICC criteria at 6 months or time to progression (TTP). Results: Pretreatment samples were available for 35 pts: 13 ER-positive and 22 ER-negative. Although responders were seen in both groups, responses were more frequently observed in ER-positive (82%) than ER-negative pts (13%). All responders expressed EGFR (median HScore 30, range 10-65) but a higher incidence of progressive disease (p=0.015) and a shorter TTP (p=0.012) were recorded in pts with high EGFR expression (clinical benefit median 36.5, range 10-160; progressive disease median 80, range 5-230). HER2 expression was similar in ER-positive TAM-R pts (54.5%) and ER-negative pts (47.6%). In ER-positive pts, high levels of HER2 did not preclude a response to gefitinib. Expression of IGF-1R did not predict response. Conclusion: Further biomarker studies, ongoing in these patients, are required to characterise predictors of response. 'Iressa' is a trademark of the AstraZeneca group of companies

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.

The tamoxifen-resistant (TAM-R) MCF-7 breast cancer cell line has been used as a model to identify the signalling pathways that enable resistant cancer cells to grow independently of steroid hormones. In TAM-R cells, peptide growth factor signalling pathways appear to be important in modified cell behaviour, growth and survival. The PI3 kinase signalling components Akt1 and Akt2 are expressed at similar levels by both parental wild-type MCF-7 and TAM-R cells, but Akt1 phosphorylation is significantly increased in TAM-R cells grown under basal conditions. High levels of basal Akt, GSK3 alpha / beta and p70S6 kinase phosphorylation are all inhibited by the PI3 kinase inhibitor, LY 294002. The ligands for the EGFR/erbB1 receptor, EGF (epidermal growth factor) and TGF alpha (transforming growth factor- alpha ) demonstrate an increased ability to activate Akt in TAM-R compared with parental MCF-7 cells and it is proposed that the preferred autocrine or paracrine activation of Akt occurs via the erbB heterodimer EGFR/erbB2 in TAM-R cells. Akt phosphorylation is reduced by gefitinib ("Iressa"/ZD1839). The results suggest that the PI3 kinase pathway plays a role in proliferation of TAM-R cells and is important in the increased EGF induced membrane ruffling detected in the resistant cells. Increased Akt1 activation may contribute to the aggressive phenotype of tamoxifen resistant ER (oestrogen receptor) positive breast cancers.

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.

We have examined the relative mRNA expression of the complement (C) regulatory proteins CD59, CD55 and CD46 in RNA isolated from 50 primary breast cancer specimens using a semiquantitative RT-PCR approach. Having normalized the mRNA expression levels of the C regulators relative to actin, we subsequently correlated their expression with estrogen receptor (ER) and various clinical, pathologic and biochemical features of the disease. CD59 and CD46 were detected in all clinical biopsies, while CD55 mRNA was detected in the majority of samples. The comparative levels of expression between the 3 regulators analyzed, using Spearman rank correlation test, revealed a significant association (p = 0.01; r = 0.36) between CD46 and CD59. CD46 exhibited the most striking pattern of association, with increased levels of expression being associated with ER-positive samples and lower levels of expression associated with a loss of differentiation and epidermal growth factor receptor positivity. Application of Spearman rank correlation test revealed CD46 expression was significantly associated with expression of ER at the level of protein (p = 0.031; r = 0.31) and mRNA (p < 0.001; r = 0.52). CD46 expression also correlated with insulin-like growth factor receptor-positive samples using Spearman rank correlation test (p = 0.016; r = 0.34), but negatively associated with tumor samples either exhibiting histologic grade 3 when compared to grades 1 or 2 or displaying elevated levels of inflammatory cell infiltrate. Immunohistochemical analysis of a limited series (n = 8) of paraffin-embedded breast cancers indicated that the level of CD46 protein expression directly associates with that of the mRNA and, where prominent, is localized in the tumor epithelial cell population, including at the plasma membrane. These data provide new information on expression of these important regulators in breast cancer and suggest that CD46 should be evaluated as a novel prognostic indicator. © 2003 Wiley-Liss, Inc.

Although many estrogen receptor-positive breast cancers initially respond to antihormones, responses are commonly incomplete with resistance ultimately emerging. Delineation of signaling mechanisms underlying these phenomena would allow development of therapies to improve antihormone response and compromise resistance. This in vitro investigation in MCF-7 breast cancer cells examines whether epidermal growth factor receptor (EGFR) signaling limits antiproliferative and proapoptotic activity of antihormones and ultimately supports development of resistance. It addresses whether the anti-EGFR agent gefitinib (ZD1839/Iressa; TKI: 1 mum) combined with the antihormones 4-hydroxytamoxifen (TAM: 0.1 mum) or fulvestrant (Faslodex; 0.1 mum) enhances growth inhibition and prevents resistance. TAM significantly suppressed MCF-7 growth over wk 2-5, reducing proliferation detected by immunocytochemistry and fluorescence-activated cell sorter cell cycle analysis. A modest apoptotic increase was observed by fluorescence-activated cell sorter and fluorescence microscopy, with incomplete bcl-2 suppression. EGFR induction occurred during TAM response, as measured by immunocytochemistry and Western blotting, with EGFR-positive, highly proliferative resistant growth subsequently emerging. Although TKI alone was ineffective on growth, TAM plus TKI cotreatment exhibited superior antigrowth activity vs. TAM, with no viable cells by wk 12. Cotreatment was more effective in inhibiting proliferation, promoting apoptosis, and eliminating bcl-2. Cotreatment blocked EGFR induction, markedly depleted ERK1/2 MAPK and protein kinase B phosphorylation, and prevented emergence of EGFR-positive resistance. Faslodex plus TKI cotreatment was also a superior antitumor strategy. Thus, increased EGFR evolves during treatment with antihormones, limiting their efficacy and promoting resistance. Gefitinib addition to antihormonal therapy could prove more effective in treating estrogen receptor-positive breast cancer and may combat development of resistance.

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.

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 α 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.

Sequential use of endocrine therapies remains the cornerstone of treatment for hormone receptor-positive advanced breast cancer, prior to use of cytotoxic chemotherapy for unresponsive disease. Fulvestrant is an estrogen receptor (ER) antagonist approved for treatment of postmenopausal women with ER+ advanced breast cancer following failure of prior antiestrogen therapy. Initially approved at a monthly dose of 250 mg, the recommended fulvestrant dose was revised to 500 mg (500 mg/month plus 500 mg on Day 14 of Month 1) following demonstration of improved progression-free survival versus fulvestrant 250 mg. We have reviewed the dose-dependent effects of fulvestrant, both from a retrospective combined analysis of dose-dependent reduction of tumor biomarkers in the pre-surgical setting (three previously reported studies: Study 18, NEWEST [Neoadjuvant Endocrine Therapy for Women with Estrogen-Sensitive Tumors] and Trial 57), and from a review of clinical studies for advanced breast cancer in postmenopausal women. Analysis of pre-surgical data revealed a consistent dose-dependent effect for fulvestrant on tumor biomarkers, with increasing fulvestrant dose resulting in greater reductions in ER, progesterone receptor and Ki67 labeling index. The dose-dependent biological effect corresponds with the dose-dependent clinical efficacy observed in the treatment of advanced breast cancer following failure of prior antiestrogen therapy. Although it remains to be determined in a Phase III trial, cross-trial comparisons suggest a dose-dependent relationship for fulvestrant as first-line treatment for advanced breast cancer. Overall, biological and clinical data demonstrate a strong dose-dependent relationship for fulvestrant, supporting the efficacy benefit seen with fulvestrant 500 mg over the 250 mg dose.

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

Objective:\ud LC-MS/MS phospho-proteomics is an essential technology to help unravel the complex molecular events that lead to and propagate cancer. We have developed a global phospho-proteomic workflow to determine activity of signaling pathways and drug targets in pancreatic cancer tissue for clinical application.\ud \ud Methods:\ud Peptides resulting from tryptic digestion of proteins extracted from frozen tissue of pancreatic ductal adenocarcinoma and background pancreas (n = 12), were labelled with tandem mass tags (TMT 8-plex), separated by strong cation exchange chromatography, then were analysed by LC-MS/MS directly or first enriched for phosphopeptides using IMAC and TiO2, prior to analysis. In-house, commercial and freeware bioinformatic platforms were used to identify relevant biological events from the complex dataset.\ud \ud Results:\ud Of 2,101 proteins identified, 152 demonstrated significant difference in abundance between tumor and non-tumor tissue. They included proteins that are known to be up-regulated in pancreatic cancer (e.g. Mucin-1), but the majority were new candidate markers such as HIPK1 & MLCK. Of the 6,543 unique phosphopeptides identified (6,284 unique phosphorylation sites), 635 showed significant regulation, particularly those from proteins involved in cell migration (Rho guanine nucleotide exchange factors & MRCKα) and formation of focal adhesions. Activator phosphorylation sites on FYN, AKT1, ERK2, HDAC1 and other drug targets were found to be highly modulated (≥2 fold) in different cases highlighting their predictive power.\ud \ud Conclusion:\ud Here we provided critical information enabling us to identify the common and unique molecular events likely contributing to cancer in each case. Such information may be used to help predict more bespoke therapy suitable for an individual case.