Therapeutic Strategy For Breast Cancer Research Articles

Abstract 187: Epigenetic memory during breast cancer progression is sustained by Smad signaling pathway

Abstract The molecular basis for the “epigenetic memory” that ensures maintenance of unique patterns of regulatory DNA methylation of selected genes responsible for cancer progression has remained elusive. Using a breast cancer progression model system, we found that hyperactive TGFβ-TGFβR-Smad2 signaling axis is involved in the maintenance of DNA hypermethylation mediated silencing of critical target genes to facilitate breast cancer progression. Disruption of TGFβ signaling caused a corresponding decrease in DNMT1 binding activity accompanied with increased expression of the target genes and acquisition of tumorigenic properties. These observations are consistent with passive demethylation of the newly synthesized DNA due to absence of sustained TGFβ-Smad signaling downstream events. In summary, we provide conclusive evidence that the reversal of the DNA hypermethylation status of gene promoters occurs as a result of TGFβ/Smad signaling pathway perturbation. By extension, we predict that inhibition of TGFβ-TGFβR-Smad2 signaling axis is an effective therapeutic strategy for breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 187.

Epigenetic Repression of PDZ-LIM Domain-containing Protein 2: IMPLICATIONS FOR THE BIOLOGY AND TREATMENT OF BREAST CANCER

The NF-kappaB transcription factor plays a pivotal role in breast cancer progression and therapy resistance. However, the mechanisms by which the tightly regulated NF-kappaB becomes constitutively activated during breast cancer pathogenesis remain obscure. Here, we report that PDZ-LIM domain-containing protein 2 (PDLIM2), an essential terminator of NF-kappaB activation, is repressed in both estrogen receptor-positive and estrogen receptor-negative breast cancer cells, suggesting one important mechanism for the constitutive activation of NF-kappaB. Indeed, PDLIM2 reexpression inhibited constitutive NF-kappaB activation and expression of NF-kappaB-targeted genes in those breast cancer cells. Importantly, PDLIM2, but not its mutants defective in NF-kappaB termination, could suppress in vitro anchorage-independent growth and in vivo tumor formation of those malignant breast cells. In addition, we have shown that PDLIM2 repression involves promoter methylation. Accordingly, treatment of the breast cancer cells with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine reverses the methylation of the PDLIM2 promoter, restored PDLIM2 expression, and suppressed tumorigenicities of human breast cancer cells both in vitro and in vivo. These studies thus provide important mechanistic insights into breast cancer pathogenesis. These studies also suggest a tumor suppression function of PDLIM2 and a therapeutic strategy for breast cancer.

Axl is an essential epithelial-to-mesenchymal transition-induced regulator of breast cancer metastasis and patient survival

Metastasis underlies the majority of cancer-related deaths. Thus, furthering our understanding of the molecular mechanisms that enable tumor cell dissemination is a vital health issue. Epithelial-to-mesenchymal transitions (EMTs) endow carcinoma cells with enhanced migratory and survival attributes that facilitate malignant progression. Characterization of EMT effectors is likely to yield new insights into metastasis and novel avenues for treatment. We show that the presence of the receptor tyrosine kinase Axl in primary breast cancers independently predicts strongly reduced overall patient survival, and that matched patient metastatic lesions show enhanced Axl expression. We demonstrate that Axl is strongly induced by EMT in immortalized mammary epithelial cells that establishes an autocrine signaling loop with its ligand, Gas6. Epiallelic RNA interference analysis in metastatic breast cancer cells delineated a distinct threshold of Axl expression for mesenchymal-like in vitro cell invasiveness and formation of tumors in foreign and tissue-engineered microenvironments in vivo. Importantly, in two different optical imaging-based experimental breast cancer models, Axl knockdown completely prevented the spread of highly metastatic breast carcinoma cells from the mammary gland to lymph nodes and several major organs and increased overall survival. These findings suggest that Axl represents a downstream effector of the tumor cell EMT that is required for breast cancer metastasis. Thus, the detection and targeted treatment of Axl-expressing tumors represents an important new therapeutic strategy for breast cancer.

Synergistic Growth Inhibition with a PI3 Kinase/mTOR Inhibitor Plus Lapatinib.

Abstract The mammalian target of rapamycin, mTOR, is a member of the phosphatidylinositol 3-kinase (PI3-K) /Akt family and plays a key role in cell growth and survival. The PI3-K/mTOR pathway is activated by many growth factor receptors in human tumor cells. In addition, the PI3-K pathway is constitutively activated by mutation in one-third of breast cancers. Therefore, blockade of the PI3-K/mTOR pathway may sensitize breast cancers to HER-2/neu inhibition. BEZ235 (Novartis Pharma AG) is a potent, oral, dual PI3-K/mTOR inhibitor currently in Phase I clinical trials. The purpose of this study was to assess the in vitro effects of combining a dual PI3-K/mTOR inhibitor (BEZ235, Novartis Pharma AG) and a lapatinib-like dual inhibitor of EGFR and HER-2/neu (GW2974) on the growth of HER-2/neu-transfected MCF-7 human breast cancer cells. Cells were exposed to the lapatinib-like compound [GW2974 (0.05-5.0µM)] or the dual PI3-K/mTOR inhibitor [BEZ235 (0.5-3nM)] alone or in combination for 3 days (12 different dose combinations were tested). Cell growth was determined using the colorimetric MTT tetrazolium dye assay. The Chou-Talalay method of isobologram analysis was performed to determine the inhibitory effect. Dose-dependent growth inhibition was observed in HER-2/neu-transfected MCF-7 cells after 3 days exposure to GW2974 (IC50: 5.0 mM) or BEZ235 (IC50: 2.2 nM). Exposure to the combination of GW2974 (0.05-0.50 mM) and BEZ235 (0.50-1.0 nM) resulted in synergistic growth inhibition in HER-2/neu-transfected MCF-7 human breast cancer cells. In summary, the combination of lapatinib plus PI3-K/mTOR warrants further investigation as a therapeutic strategy for breast cancer. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 6114.

Growth Inhibition of Human Breast Cancer Cells with an Iron Chelator (Deferasirox) and Docetaxel.

Abstract Treatment of cells with an iron chelator results in G1/S arrest and apoptosis. Tumor cells appear to be more sensitive to iron chelation probably because they express higher levels of the iron containing enzyme, ribonucleotide reductase. Previously, our laboratory has shown that an iron chelator (deferoxamine) inhibited the growth of a variety of cancer cell lines and also prolonged survival in tumor bearing mice. Docetaxel is an antineoplastic agent belonging to the taxane family. Docetaxel induces bcl-2 phosphorylation and subsequent apoptosis and is effective in the treatment of patients with breast cancer. The purpose of this study was to assess the in vitro effects of combining an iron chelator, deferasirox (Exjade: Novartis Pharma AG), and docetaxel on the growth of MCF-7 and HER-2/neu-transfected MCF-7 human breast cancer cells. Cells were exposed to deferasirox or docetaxel alone or in combination for 3 days (9 different dose combinations were tested). Cell growth was determined using the colorimetric MTT tetrazolium dye assay. Dose-dependent growth inhibition was observed in both the MCF-7 and the HER-2/neu-tranfected MCF-7 cells after 3 days exposure to deferasirox (IC50: 1.5 and 2.3 μM, respectively) or docetaxel (IC50: 0.9 nM and 2.0 nM, respectively). Exposure to the combination of deferasirox (0.25 – 1.5 μM) and docetaxel (0.1 – 0.5 nM) resulted in synergistic growth inhibition in both the MCF-7 and HER-2/neu-transfected MCF-7 human breast cancer cells. Sequencing experiments with the HER-2/neu-transfected MCF-7 cells showed that simultaneous exposure of the cells to deferasirox and docetaxel was superior to pretreating the cells with either of the agents 6 or 24 hours prior to addition of the second agent. These results suggest this combination warrants further investigation as a therapeutic strategy for breast cancer. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 5075.

Preliminary Study of Pre-Metastatic Niche (PMN) in Nonmetastatic Nodes for the Assessment of Metastasis Risk in Breast Cancer (BC).

Abstract Bone marrow-derived hematopoietic progenitor cells (HPCs) seem to participate in early spread of cancer cells by forming a peculiar and highly organized micro-environment called PMN. Identifying such HPCs in the PMNs is emerging as a key step in assessing risk of metastasis.In this preliminary study we analysed 294 axillar nodes (271 pN0 and 23 pN1a) obtained from 30 pts undergone surgery for breast ductal invasive carcinoma in the period 1998-2000.Pts mean age was 61.2 years (range 45-79); 9 pts (30%) were premenopausal and 21 (70%) postmenopausal; 13 pts (43.3%) had ER+, 10 (33.3%) PR+; MIB1 was ≤ 25% in 22 pts (73.3%), >25% in 6 (20%), unknown in 2 pts. After a median follow-up of 81.27 mos (range 12–136), 14 pts are disease-free, 9 pts dead of metastatic disease. Involved sites were: 1 loco-regional, 7 bone, 3 lung, 4 liver, 1 brain.Samples were fixed in 10% neutral buffered formalin for 12-72 hrs and included in paraffin at 56°C; 4 mm thick sections were pre-treated in microwave owen in 10 mM citric acid, pH 6.0 and incubated overnight with the following poly/mono-clonal antisera: VEGF-R1 (Santa Cruz Biothechnology 1:400),CD 133(Abgent 1:80),CD 117 (DAKO 1:500),CD-34 (DAKO 1:50).Clusters of immunoreactive cells were evident with CD 117 and CD 34 in all nodes.A different degree of immunopositivity was found with VEGR-R1 and CD 133 antibodies for different sites of metastasis; in particular, the cases spread in lungs, liver and central nervous system were more immunoreactive, compared to those with bone localization. All nodes classified as pN1a showed no immunoreactivity.The prognostic significance of HPCs in PMN needs to be further investigated, especially in node-negative BCs. The immunomorphologic aspect of the lymph node microenvironment could have significant clinical implications, in order to optimize BC therapeutic strategies. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 6024.

Abstract B105: Axl is an essential epithelial-to-mesenchymal transition-induced regulator of breast cancer metastasis and patient survival

Abstract Metastasis underlies the majority of cancer-related deaths. Hence, furthering our understanding of the molecular mechanisms that enable tumor cell dissemination is a vital health issue. Epithelial-to-mesenchymal transitions (EMT) endow carcinoma cells with enhanced migratory and survival attributes that facilitate malignant progression. Characterization of EMT effectors is likely to yield new insights into metastasis and novel avenues for treatment. We show that the presence of the receptor tyrosine kinase Axl in mammography-detected primary breast cancers independently predicts strongly reduced overall patient survival, and matched patient metastatic lesions show enhanced Axl expression. We demonstrate that Axl is strongly induced by epithelial-to-mesenchymal transition in pre-malignant mammary epithelial cells that establishes an autocrine signaling loop with its ligand, Gas6. Using epi-allelic RNA interference analysis in metastatic breast cancer cells we delineated a distinct threshold of Axl expression for mesenchymal-like in vitro cell invasiveness, and to form tumors in foreign and tissue engineered microenvironments in vivo. Importantly, Axl knockdown completely prevented the spread of highly metastatic breast carcinoma cells from the mammary gland to lymph nodes and several major organs, and increased overall survival, in two different optical imaging-based experimental breast cancer models. Thus, Axl represents a novel downstream effector of tumor cell EMT that is required for breast cancer metastasis. The detection and targeted treatment of Axl-expressing tumors represents an important new therapeutic strategy for breast cancer. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B105.

Current issues and perspectives on breast cancer diagnosis

Increased incidence of breast cancer and new developments in breast cancer therapy in Japan have led to changes in procedures for the pathological diagnosis of the breast. There have been changes in the biopsy process from the conventional incisional or excisional biopsy to needle core biopsy, which acquires only minute samples, in which most pathologists currently have little experience. The diagnostic process has also seen advances because of the adoption of new methodologies, such as the immunohistochemistry of hormone receptors and HER2, not to mention the concept of triple-negative breast cancer in addition to the conventional benign–malignant diagnosis. It can therefore be said that there is a host of opportunities for tackling these new technologies. This special edition provides commentaries from the pathologist’s viewpoint on the following subjects: (1) therapeutic strategies for breast cancer based on histological type; (2) the usefulness of immunohistochemistry on the differential diagnosis between benign and malignant breast tumor; (3) morphological characteristics of the basal-like subtype of breast carcinoma with special reference to cytopathological features; (4) gene and chromosomal alterations in sporadic breast cancer: correlation with histopathological features and implication for genesis and progression; (5) a new reporting form for breast cytology. All these reports, which feature the latest cutting-edge knowledge and concepts, come from sites where research activities are focused chiefly on the pathological diagnosis of breast cancer. Having been in charge of planning and editing, I will be more than happy if this special edition makes a contribution to clinical and pathological research centered on the breast.

Therapeutic strategies for breast cancer based on histological type

Breast cancer has various histological types that reflect not only morphological features but also biological characteristics. Therefore, it is not an exaggeration to say that breast cancers of different histological types are different diseases. It is generally accepted that the histological types of breast cancer are clinically significant because they serve as prognosticators and as the common language for improving the accuracy of clinical diagnosis. It is necessary to diagnose breast cancer at the level of not only histological findings by needle biopsy, but also the histologic type based on diagnostic imaging and cytological diagnosis. From the viewpoint of treatment, preoperative drug therapy is being performed more frequently to shrink tumors before breast-conserving therapy or to determine treatment sensitivity. The prognosis is favorable for patients who respond completely or patients in whom interstitial infiltration is completely eliminated histopathologically, and, as a result, it is important to assess therapeutic efficacy clinically and pathologically. Past experience has shed some light on differentiating cancers responsive to drug therapy from those unresponsive to drug therapy, as well as differentiating cancers in which therapeutic efficacy can be easily ascertained from those in which therapeutic efficacy cannot be easily ascertained. Preoperative drug therapy can be planned by making a histological diagnosis based on needle biopsy findings. Preoperative drug therapy is not indicated for noninvasive carcinoma and papillotubular carcinoma (invasive carcinoma with predominant intraductal components). While complete loss of interstitial infiltration can be expected with solid-tubular carcinoma, it cannot be expected with other histological types, such as invasive lobular carcinoma, adenoid cystic carcinoma, or metaplastic carcinoma (squamous-cell carcinoma and spindle-cell carcinoma). On therapeutic response assessment, the clinical and pathological findings generally match for solid-tubular carcinoma but not for scirrhous carcinoma and invasive lobular carcinoma. With mucinous carcinoma, mucus accumulation can remain, even though most cancer cells disappear; as a result, assessment based on tumor diameter changes is difficult. Histological diagnosis is also significant from the viewpoint of drug sensitivity, and it is important to maintain the accuracy of histological diagnosis.

Sensitivity of breast cancer cell lines to the novel insulin-like growth factor-1 receptor (IGF-1R) inhibitor NVP-AEW541 is dependent on the level of IRS-1 expression

To investigate the potential value of targeting insulin-like growth factor-1 receptor (IGF-1R) in breast cancer, we examined the effects of NVP-AEW541, a selective small-molecule inhibitor of the IGF-1R tyrosine kinase, in a panel of 16 breast cancer cell lines. All cell lines expressed IGF-1R, but MCF-7 expressed much higher levels of insulin receptor substrate-1 (IRS-1) than the others. NVP-AEW541 was more potent at inhibiting growth of MCF-7 cells as compared to the others (IC 50, 1 μM vs. ≈7 μM). Comparing MCF-7 to T47D cells, which express IGF-1R at a level identical to MCF-7 but have less than 1/30 the amount of IRS-1, NVP-AEW541 caused cell-cycle arrest at the G1–S boundary, reduced in vitro cell migration, and enhanced the cytotoxic effects of vinorelbine and paclitaxel in MCF-7, but not in T47D. While NVP-AEW541 decreased the phosphorylation of IGF-1R in both cell lines, it inhibited phosphorylation of Akt and disrupted the IRS-1/PI3 K complex only in MCF-7. These findings suggest that inhibiting IGF-1R may be an effective therapeutic strategy for breast cancers that co-express IGF-1R and IRS-1 at high levels.

Novel therapeutic strategy for breast cancer: mammalian target of rapamycin inhibition

Background: Mammalian target of rapamycin (mTOR) plays a central role in regulating cellular protein synthesis. Dysregulation of mTOR signaling pathway is strongly associated with tumorigenesis, angiogenesis, tumor progression and drug resistance. Inhibition of mTOR might not only promote cell cycle arrest, but also sensitize resistant cancer cells to chemotherapeutic and other targeted agents. Objective: To review and summarize the mechanism of mTOR on regulation of protein synthesis and latest clinical data, and to discuss the novel therapeutic strategy for the use of mTOR inhibitors in the treatment of breast cancer. Methods: A review of published literatures and conference abstracts obtained from MEDLINE, American Society of Clinical Oncology Meeting and San Antonio Breast Cancer Symposia proceedings for results of previous preclinical and latest clinical studies of mTOR inhibition in breast cancer was performed. Conclusions: mTOR inhibitors seemed to be potentially useful for the treatment of breast cancer with acceptable safety profile. The challenge remains the identification of suitable candidates with different phenotypes. More structured studies incorporating molecular, clinical and translational research need to be initiated. Future research on mTOR inhibitors for breast cancer should focus on the evaluation of optimal schedule, patient selection and combination strategies to maximize the use of this new class of targeted agents.

Role Of Membrane Cholesterol Content In The Activity Of Cyclooxygenase-2 (COX-2) In MCF-7 Human Breast Cancer Cells

Cyclooxygenase-2 (COX-2) and its product PGE2 are known to increase both angiogenesis and resistance to apoptosis (promoting tumor growth) and to enhance the penetration of cancer cells into adjacent tissues (causing metastasis). Thus, knowing how the activity of COX2 is regulated at the cellular level has implications for breast cancer therapeutic strategies. The goal of this research is to unravel a new molecular mechanism for regulating the activity of COX-2. The proposed molecular mechanism may be elucidated by using the sterol superlattice model. In plasma membranes, arachidonic acid (AA) is released by phospholipase A2 (PLA2). Cyclooxygenase then converts AA to prostaglandins (e.g., PGE2). The activity of PLA2 is known to vary with membrane cholesterol content in an alternating manner, showing a local minimum at critical sterol mole fractions (Cr) for maximal superlattice formation. Hence, it is logical to hypothesize that the activity of COX (including the isoform COX-2) also varies with cholesterol content in a biphasic manner. In this study, the cholesterol content in MCF-7 human breast cancer cells was decreased systematically by using methyl-beta-cyclodextrin. A biphasic change in COX-2 activity, as monitored by the Cayman COX-2 assay with minor modifications, was observed at certain cell cholesterol content Crcell. The cholesterol content near Crcell could serve as a fine-tuning mechanism to regulate COX-2 activity and PGE2 production, and consequently, cancer cell growth and metastasis. (supported by DOD, NSF and PDOH)

Breast tumor cells with PI3K mutation or HER2 amplification are selectively addicted to Akt signaling.

Abstract Abstract #3061 Background: Activation of the PI3K/Akt pathway is a common event in human cancers and is responsible for key aspects of the transformed phenotype. This pathway is activated in most breast cancers, but by different mechanisms including HER2 amplification, PI3K mutation and PTEN inactivation. Inhibition of this pathway is thus thought to be clinically useful, but the functional output of PI3K/Akt signaling and the corresponding clinical effects of its inhibition may be due to the mechanisms by which it is activated. The objective of this study was to determine the role of Akt activation in breast cancer as a function of mechanism of activation and whether inhibition of Akt signaling is a feasible approach to therapy.
 Methodology/Principal Findings: Using a panel of human breast cancer cell lines that harbor HER2 amplification, PI3K or PTEN mutations, or wild-type of PI3K and PTEN, and a selective, PH-domain dependent, allosteric inhibitor of Akt1 and Akt2 (AKTi-1/2), and a variety of biochemical, molecular, and cellular techniques as well as in vivo xenograft models, we show that AKTi-1/2 resulted in the selective inhibition of breast tumor cell lines with either mutant PI3K or amplified HER2, but not those in which PI3K/Akt signaling is not mutationally activated. Cellular sensitivity was due to induction of G1 arrest and of apoptosis and associated with inhibition of Akt signaling, loss of D-cyclin expression, and dephosphorylation of Rb. Most importantly, AKTi-1/2 effectively inhibited Akt kinase and its downstream effectors in vivo and caused complete suppression of the growth of breast cancer xenografts with PI3K mutation or HER2 amplification, including models of the latter selected for resistance to Herceptin. Furthermore, chronic administration of the drug was well-tolerated, causing only transient hyperglycemia without gross toxicity to the host despite the pleiotropic normal functions of Akt.
 Conclusions/Significance: Our data demonstrate the exquisite dependence of PI3K-mutated or HER2-amplified breast cancer cells on Akt signaling, and that effective inhibition of Akt in tumors is feasible and effective in vivo. These findings suggest that direct inhibition of Akt may represent a therapeutic strategy for breast cancers that are addicted to the pathway and in patients with clinical resistance to Herceptin. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 3061.

Targeting EGFRs and Src signaling with a modified ectodomain of human EGFR (EBIP) and dasatinib in breast cancer.

Abstract Abstract #3069 It is becoming increasingly evident that many solid tumors including breast cancer show increased activation of several growth factor receptors, specifically EGFR and its family members (EGFRs) as well as c-Src, a non-receptor tyrosine kinase that promote proliferation, inhibit apoptosis and induce metastasis. The EGF-receptor (EGFR) consists of an extracellular ligand-binding domain, a hydrophobic transmembrane region, and an intracellular tyrosine kinase domain. The extracellular or ectodomain of EGFR is essential for ligand-binding and subsequent homo/heterodimerization of the receptor. This raises the possibility that the ectodomain of EGFR could be utilized to inhibit EGFR functions and in turn cellular growth. Recently, we generated an ectodomain of human EGFR (hEGFR), composed of 1-448 amino acids fragment of hEGFR and the 30 amino acid epitope (known as “U” region) of rat EGFR-Related Protein (ERRP) fused at the carboxy-terminal end. The new protein, referred to as EBIP (ErbB Inhibitory Protein), as has been observed for ERRP (Xu et al. Mol. Cancer Ther. 2005 (4): 435-42), inhibits growth of colon and breast cancer cells that express varying levels of EGFR and/or its family members. We hypothesize that inhibition of c-Src and EGFRs will be an effective therapeutic strategy for breast cancer. To test our hypothesis we used Dasatinib (BMS-354825; Bristol-Myers Squibb), a newly developed highly potent, ATP-competitive Src and Abl kinase inhibitor and ErbB Inhibitory Protein (EBIP). Four different breast cancer cell lines (MDA-MB-468, SKBr-3, MDA-MB-453 and MDA-MB-231) expressing different levels of EGFR and/or its family members were used. Both EBIP and dasatinib caused a dose dependent inhibition of growth in all breast cancer cells, but dasatinib was less effective compared to EBIP. However, the combination of Dasatinib and EBIP synergistically inhibited cell growth. In EGFR overexpressing MDA-MB-468 cells, the combination therapy also inhibited invasion through extracellular matrix and decreased colony formation. Induction of apoptosis could be attributed to activation of caspases -9 and -8 and by modulating phosphorylation of anti- and pro-apoptotic proteins, Bcl-2 and Bad, respectively. The combination of Dasatinib and EBIP produced a greater inhibition of EGFR and Src activation and their downstream signaling pathways involving Akt and p44/42 MAPK. Additionally, we observed that EGFR promoter activities as well as transactivation of NFκB in MDA-MB-468 cells were greatly reduced. In conclusion, our data suggest that combination treatment of Dasatinib and EBIP that target EGFRs and Src could be a highly effective therapy for breast cancer than mono therapy, as they are likely to impact several aspects of tumor progression. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 3069.

Patterns of recurrence of early breast cancer according to estrogen receptor status: a therapeutic target for a quarter of a century.

The current therapeutic strategy in breast cancer is to identify a target, such as estrogen receptor (ER) status, for tailoring treatments. We investigated the patterns of recurrence with respect to ER status for patients treated in two randomized trials with 25 years' median follow-up. In the ER-negative subpopulations most breast cancer events occurred within the first 5-7 years after randomization, while in the ER-positive subpopulations breast cancer events were spread through 10 years. In the ER-positive subpopulation, 1 year endocrine treatment alone significantly prolonged disease-free survival (DFS) with no additional benefit observed by adding 1 year of chemotherapy. In the small ER-negative subpopulation chemo-endocrine therapy had a significantly better DFS than endocrine alone or no treatment. Despite small numbers of patients, "old-fashioned" treatments, and competing causes of treatment failure, the value of ER status as a target for response to adjuvant treatment is evident through prolonged follow-up.

Crosstalk Between IGF1R and Estrogen Receptor Signaling in Breast Cancer

After the discovery that depriving certain breast tumors of estrogen promoted tumor regression, therapeutic strategies aimed at depriving tumors of this hormone were developed. The tumorigenic properties of estrogen are regulated through the estrogen receptor-alpha (ER), making understanding the mechanisms that activate this receptor highly relevant. In addition to estrogen activating the ER, other growth factor pathways, such as the insulin-like growth factors (IGFs), can activate the ER. This review will examine the interaction between these two pathways. Estrogen can activate the growth stimulatory properties of the IGF pathway via ER's genomic and non-genomic functions. Further, blockade of ER function can inhibit IGF-mediated mitogenesis and blocking IGF action can inhibit estrogen stimulation of breast cancer cells. Collectively, these observations suggest that the two growth regulatory pathways are tightly linked and a more thorough understanding of the mechanism of this crosstalk could lead to improved therapeutic strategies in breast cancer.

Human X‐Box binding protein‐1 confers both estrogen independence and antiestrogen resistance in breast cancer cell lines

Human X-box binding protein-1 (XBP1) is an alternatively spliced transcription factor that participates in the unfolded protein response (UPR), a stress-signaling pathway that allows cells to survive the accumulation of unfolded proteins in the endoplasmic reticulum lumen. We have previously demonstrated that XBP1 expression is increased in antiestrogen-resistant breast cancer cell lines and is coexpressed with estrogen receptor alpha (ER) in breast tumors. The purpose of this study is to investigate the role of XBP1 and the UPR in estrogen and antiestrogen responsiveness in breast cancer. Overexpression of spliced XBP1 [XBP1(S)] in ER-positive breast cancer cells leads to estrogen-independent growth and reduced sensitivity to growth inhibition induced by the antiestrogens Tamoxifen and Faslodex in a manner independent of functional p53. Data from gene expression microarray analyses imply that XBP1(S) acts through regulation of the expression of ER, the antiapoptotic gene BCL2, and several other genes associated with control of the cell cycle and apoptosis. Testing this hypothesis, we show that overexpression of XBP1(S) prevents cell cycle arrest and antiestrogen-induced cell death through the mitochondrial apoptotic pathway. XBP1 and/or the UPR may be a useful molecular target for the development of novel predictive and therapeutic strategies in breast cancer.

Critical role of FoxO3a transcription factor in response to therapy with EGFR inhibitors, gefitinib and lapatinib, in breast cancer

21018 Background: Inhibition of the EGF-receptor is a potential therapeutic strategy for breast cancer. The PI3-K/PKB signalling pathway plays an important role in cell proliferation, survival and malignant transformation. The Forkhead box group O transcriptional factor, FoxO3a, is a direct downstream target of PKB. Here we studied the role of the FoxO3a in response to the EGFR inhibitors Gefitinib and Lapatinib. Methods: Response of breast cancer cell lines to treatment with Gefitinib and Lapatinib was evaluated by proliferation assay and FACS analysis. Expression of FoxO3a and its downstream targets was analysed using Western blotting. Sub-cellular localisation of FoxO3a was analysed by confocal microscopy. FoxO3a mRNA level was measured by RTq-PCR. siRNA transfections were performed using Oligofectamine reagent. Immunohistological staining was performed on patient samples to validate our findings. Results: Treatment of a panel of breast cancer cell lines with both inhibitors resulted in decreased proliferation due to G1-arrest and apoptosis in two sensitive cell-lines, BT474 and SKBR3. Western blot analysis revealed that response to the treatment was associated with a decrease in PKB and FoxO3a phosphorylation and thus the nuclear relocalisation of FoxO3a. Confocal microscopy confirmed that treatment induced nuclear translocation of FoxO3a only in sensitive but not resistant cells. This relocation of FoxO3a was accompanied by an induction of FoxO3a mRNA and its principal targets, p27kip1 and Bim. Transfection of the sensitive BT474 cells with FoxO3a specific siRNA resulted in a significant reduction in FoxO3a protein expression and Gefitinib-induced cell death. Comparison of immunohistochemical staining of biopsies from breast cancer patients before and after Gefitinib treatment revealed that therapy resulted in a significant increase in nuclear FoxO3a staining. Conclusion: Our results demonstrate for the first time that the EGFR-family inhibitors, Gefitinib and Lapatinib, specifically target FoxO3a to induce cell cycle arrest and apoptosis. This finding helps to define the mechanism of action of Gefitinib and Lapatinib and may provide novel insights into the molecular basis for resistance to these inhibitors. No significant financial relationships to disclose.

Requirement of Pygopus 2 in breast cancer.

The development of novel therapeutic strategies for breast cancer requires the identification of molecular targets involved in malignancy. Human Pygopus (Pygo)-1 and -2 are recently discovered components of the Wnt signaling pathway required for beta-Catenin/Tcf dependent transcription in embryos and colorectal cancer cells, but the role of these proteins in malignant cell growth and survival has not yet been determined. We report the expression and requirement for proliferation of hPygo2 in breast cancer cells. hPygo2 protein was overexpressed in malignant breast tumors and in the nuclei of five breast cancer cell lines, but was not expressed in the nuclei of non-malignant breast cells. Phosphorothioated antisense oligonucleotides were used to specifically knockdown expression hPygo2 in Mcf-7 and MDA-MB-468 cell lines. hPygo2 was required for the growth, in tissue culture and anchorage-independent assays, of both cell lines and for the expression of the Wnt target gene Cyclin D1. We conclude that hPygo2 is highly expressed in, and required for the growth of breast carcinoma cells.

Mechanisms of endocrine resistance and novel therapeutic strategies in breast cancer

Tamoxifen has been the mainstay of hormonal therapy in both early and advanced breast cancer patients for approximately three decades. The availability of novel compounds such as aromatase inhibitors (AIs) and fulvestrant, with different mechanism of action, is changing the scenario of endocrine treatment of postmenopausal breast cancer patients. In this review article, we have summarized the current knowledge of the mechanisms of resistance to endocrine therapy, in order to derive information that might be useful for therapeutic intervention. We propose that resistance to endocrine therapy is a progressive, step-wise phenomenon induced by the selective pressure of hormonal agents, which leads breast cancer cells from an estrogen-dependent, responsive to endocrine manipulation phenotype to a non-responsive phenotype, and eventually to an estrogen-independent phenotype. In particular, evidence suggests for each 'action' introduced to block estrogen stimulation of breast cancer cells (i.e. treatment with anti-estrogen), there are one or more corresponding 'reactions' that tumor cells can use to escape our attempts to block their growth: estrogen hypersensitivity associated with increased transcriptional activity of estrogen receptor alpha (ERalpha) and/or increased non-genomic activity of ERalpha, estrogen supersensitivity, increased growth factor signaling, suppression of ERalpha expression and finally estrogen independence. Activation of growth factor signaling is involved in each step of this phenomenon, and might ultimately substitute estrogen in sustaining the growth and the survival of breast cancer cells. In this respect, results of pre-clinical and clinical studies with AIs, fulvestrant and signaling inhibitors sustain this hypothesis. More importantly, the knowledge of the mechanisms involved in the resistance of breast cancer cells to endocrine therapy offers potential for novel therapeutic strategies.