Herceptin: A First Assault on Oncogenes that Launched a Revolution.

This year's Lasker Clinical Research Award honors H. Michael Shepard, Dennis J. Slamon, and Axel Ullrich for their invention of Herceptin, the first monoclonal antibody that blocks a cancer-causing protein, and for its development as a life-saving therapy for women with breast cancer.

Subcellular distribution and mechanism of action of AL906, a novel and potent EGFR inhibitor rationally designed to be green fluorescent.

To enhance the potency of EGFR inhibitors, we developed a novel strategy that seeks to conjugate EGFR to a bioactive moiety leading to a molecule termed "combi-molecule". In order to mimic the penetration of this type of molecules, based upon previously reported structure activity relationship studies, we designed a new molecule containing a quinazoline moiety tethered to a p-nitrobenzoxadiazole (NBD) moiety [molecular weight (MW) 700]. Despite its size, AL906 growth inhibitory activity was superior to that of the clinical drug gefitinib. Furthermore, AL906 retained significant EGFR inhibitory activity and good cellular penetration with abundant distribution in the perinuclear region of the cells. In an isogenic NIH3T3 transfected cell panel, it selectively inhibited the growth  of the NIH3T3-EGFR and HER2 transfectants. Confocal microscopy analysis revealed that it was capable of penetrating multilayer aggregates although to a lesser extent than FD105, a small inhibitor of EGFR inhibitor of the same class (MW 300). Its ability to inhibit EGFR auto-phosphorylation in monolayer culture was stronger than in the aggregates. The results suggest that our strategy did not negatively affect EGFR inhibitory potency, EGFR selectivity and growth inhibition. However, its molecular size may account for its decreased aggregate penetration when compared with a smaller EGFR inhibitor of the quinazoline class.

Rapid screening of drug candidates against EGFR/HER2 signaling pathway using fluorescence assay.

Over the recent decade, the calcium-based assays have gained much popularity in order to discover new drugs. Since breast cancer is the second cause of death in the female population, rapid and effective methods are needed to screen drug compounds with fewer side effects. Human epidermal growth factor receptor 2 (HER2) increases intracellular free Ca2+ on its signaling pathways. In the present study, BT474 cell line, which overexpresses HER2 receptor, was selected and using fura-2-AM, intracellular Ca2+ release was investigated. The changes in the concentration of intracellular Ca2+ were evaluated by variation in the amount of fluorescence intensity. In the presence of epidermal growth factor (EGF), an increase in fluorescence intensity was observed so that after 20 min it raised to the maximum level. After treatment of BT474 cells by lapatinib, as a tyrosine kinase inhibitor (TKI), the signaling pathway of EGFR/HER2 heterodimer was significantly inhibited, which resulted in a decrease in Ca2+ entry into the cytoplasm and fluorescence emission decreased. The IC50 value for the effect of lapatinib on BT474 cells was 113.2 nmol/L. Our results suggest this method is a simple, efficient and specific approach and can potentially be useful for screening new drug candidates against EGFR/HER2 heterodimer signaling pathways. Graphical abstract ᅟ.

A Nanobody Activation Immunotherapeutic that Selectively Destroys HER2-Positive Breast Cancer Cells.

We report a rationally designed nanobody activation immunotherapeutic that selectively redirects anti-dinitrophenyl (anti-DNP) antibodies to the surface of HER2-positive breast cancer cells, resulting in their targeted destruction by antibody-dependent cellular cytotoxicity. As nanobodies are relatively easy to express, stable, can be humanized, and can be evolved to potently and selectively bind virtually any disease-relevant cell surface receptor, we anticipate broad utility of this therapeutic strategy.

Novel cancer therapies: treatments driven by tumour biology.

The traditional view is that treatments within oncology largely consist of chemotherapy, which aims to maximise damage to the rapidly dividing cancer cells but often at the expense of normal cells and overall quality of life for the patient. The development of anticancer drugs has changed from the serendipitous discoveries of the past, to today's purposeful targeting of cancer cells which takes advantage of novel technological developments and a greater understanding of tumour biology. The aim of these new treatments is to affect the essential function of the cancer cell while sparing normal cells, and limiting side effects. The phenotypic characteristics of tumours, such as unregulated growth signalling, development of new vascular systems and the evasion of immune destruction are used to identify potential drug targets. Here we review the clinical evidence and molecular mechanisms for novel therapies that are currently in use and those that are in development.

Specific apoptosis induction by the dual PI3K/mTor inhibitor NVP-BEZ235 in HER2 amplified and PIK3CA mutant breast cancer cells.

NVP-BEZ235 is a dual PI3K/mTOR inhibitor currently in phase I clinical trials. We profiled this compound against a panel of breast tumor cell lines to identify the patient populations that would benefit from such treatment. In this setting, NVP-BEZ235 selectively induced cell death in cell lines presenting either HER2 amplification and/or PIK3CA mutation, but not in cell lines with PTEN loss of function or KRAS mutations, for which resistance could be attributed, in part to ERK pathway activity. An in depth analysis of death markers revealed that the cell death observed upon NVP-BEZ235 treatment could be recapitulated with other PI3K inhibitors and that this event is linked to active PARP cleavage indicative of an apoptotic process. Moreover, the effect seemed to be partly independent of the caspase-9 executioner and mitochondrial activated caspases, suggesting an alternate route for apoptosis induction by PI3K inhibitors. Overall, this study will provide guidance for patient stratification for forthcoming breast cancer phase II trials for NVP-BEZ235.

Review of hormonal treatment of breast cancer.

This critical review focuses on the role of steroid hormones and their receptors in the development and treatment of breast cancer, with special reference to estrogen receptors, as well as mechanisms of receptor-ligand interactions, response or resistance to hormonal therapy against breast cancer, in conjunction with other modalities like surgery and chemotherapy. Tamoxifen is used in hormonal treatment of breast cancer for up to five years, depending on the presentation. However, there have been recent developments in hormonal therapy of breast cancer in the last ten years, with the introduction of many different alternative therapies for this condition. A critical review of published articles in Pubmed/Medline, Athens, AJOL, NHS Evidence, Science Direct and Google, relating to hormonal treatment of breast cancer, was undertaken, in order to evaluate the mechanisms of estrogen receptor-ligand interactions, their involvement in the etio-pathogenesis of breast cancer, resistance of breast cancer cells to anti-hormonal agents, as well as ways of treating breast cancer using anti-hormone drugs like tamoxifen. Although tamoxifen is the established drug for hormonal treatment of breast cancer, cases of hormone resistance breast cancer have been described recently in the literature. This can happen from the beginning, or during treatment. Therefore, we aim to examine the causes of resistance to hormonal treatment with a view to understand the options of tackling this problem, and suggest other novel alternative hormonal therapies that can be tried, which may overtake tamoxifen in the future. We also seek to emphasize that hormonal therapy has a definite place in the treatment of breast cancer along with surgery, chemotherapy and radiotherapy, as the disease is often considered to be multi-systemic even from the beginning.

Geldanamycin-induced down-regulation of ErbB2 from the plasma membrane is clathrin dependent but proteasomal activity independent.

ErbB2, a member of the epidermal growth factor receptor family, is overexpressed in a number of human cancers. In contrast to the epidermal growth factor receptor, ErbB2 is normally endocytosis resistant. However, ErbB2 can be down-regulated by inhibitors of heat shock protein 90, such as geldanamycin. We now show that geldanamycin induces endocytosis and lysosomal degradation of full-length ErbB2. We further report that the endocytosis of ErbB2 is dynamin and clathrin dependent. When ErbB2 was retained at the plasma membrane due to knockdown of clathrin heavy chain, the intracellular part of ErbB2 was degraded in a proteasomal manner. However, our data strongly suggest that proteasomal activity is not required for geldanamycin-induced endocytosis of ErbB2 in SKBr3 cells. Interestingly, however, proteasomal inhibitors retarded degradation of ErbB2, and electron microscopy analysis strongly suggested that proteasomal activity is required to sort internalized ErbB2 to lysosomes. Because geldanamycin derivatives and inhibitors of proteasomal activity are both used in experimental cancer treatment, knowledge of molecular mechanisms involved in geldanamycin-induced down-regulation of ErbB2 is important for future design of cancer treatment.

Freund's vaccine adjuvant promotes Her2/neu breast cancer.

BACKGROUND: Inflammation has been linked to the etiology of many organ-specific cancers. Indirect evidence suggests a possible role for inflammation in breast cancer. We investigated whether the systemic inflammation induced by Freund's adjuvant (FA) promotes mammary carcinogenesis in a rat model in which cancer is induced by the neu oncogene. METHODS: The effects of FA on hyperplastic mammary lesions and mammary carcinomas were determined in a neu-induced rat model. The inflammatory response to FA treatment was gauged by measuring acute phase serum haptoglobin. In addition, changes in cell proliferation and apoptosis following FA treatment were assessed. RESULTS: Rats receiving FA developed twice the number of mammary carcinomas as controls. Systemic inflammation following FA treatment is chronic, as shown by a doubling of the levels of the serum biomarker, haptoglobin, 15 days following initial treatment. We also show that this systemic inflammation is associated with the increased growth of hyperplastic mammary lesions. This increased growth results from a higher rate of cellular proliferation in the absence of changes in apoptosis. CONCLUSION: Our data suggests that systemic inflammation induced by Freund's adjuvant (FA) promotes mammary carcinogenesis. It will be important to determine whether adjuvants currently used in human vaccines also promote breast cancer.

Breast cancer biomarkers and HER2 testing after 10 years of anti-HER2 therapy.

The HER2 (ERBB2) oncogene encodes a transmembrane tyrosine kinase receptor that has evolved as a major biomarker of invasive breast cancer and target of therapy for the disease. HER2 gene amplification or protein overexpression is encountered in approximately 20% of newly diagnosed breast cancers and is a validated adverse prognostic factor with a mean relative risk for overall survival of 2.74. A series of quality assurance recommendations for the marketed slide-based HER2 testing approaches including immunohistochemistry (IHC), fluorescence in situ hybridization (FISH) and chromogenic in situ hybridization (CISH/SISH) has recently been published by the American Society of Clini- cal Oncology - College of American Pathologists (ASCO-CAP). Testing issues, such as the impact of chromosome 17 polysomy and local versus central HER2 testing and emerging novel HER2 testing techniques, including messenger RNA (mRNA)-based testing by real time polymerase chain reaction (RT-PCR) and DNA microarray methods, HER2 receptor dimerization, phosphorylated HER2 receptors and HER2 status in circulating tumor cells are of current interest in the management of breast cancer. Also of significant interest are the evolving lists of biomarkers proposed to predict resistance to the major anti-HER2 therapies, trastuzumab and lapatinib.

Drug Insight: intracellular inhibitors of HER2--clinical development of lapatinib in breast cancer.

Targeting the human epidermal growth factor receptor type 2 (HER2) in breast cancer patients whose tumors overexpress HER2 has been clearly demonstrated to be effective in clinical trials with the monoclonal antibody trastuzumab. Not all patients, however, respond to trastuzumab therapy. Lapatinib is an oral receptor tyrosine kinase inhibitor that targets HER2 and the EGFR. Preclinical data reveal that lapatinib has activity in trastuzumab-resistant cell lines as well as synergistic activity with trastuzumab. In a pivotal phase III trial, a combination of lapatinib and capecitabine significantly decreased the risk of disease progression relative to capecitabine alone in women with HER2-positive advanced or metastatic breast cancer previously treated with anthracyclines, taxanes, and trastuzumab. Other trials are evaluating lapatinib in inflammatory breast cancer--for which encouraging data have been reported--in combination with hormone therapy, in combination with trastuzumab, and as an adjunct to adjuvant therapy for early-stage disease. Notably, lapatinib has not been associated with serious or symptomatic cardiotoxicity in clinical trials. It can cross the blood-brain barrier and might therefore have a role in preventing central-nervous-system progression. These features make lapatinib an ideal agent to evaluate more fully in HER2-positive metastatic and early-stage breast cancer.

European oncologists' preferences for the management of breast cancer: case presentations and expert commentary.

The development of new cytotoxic and biological agents has brought a welcome extension in the range of therapeutic options available to women with both early-stage and advanced breast cancer. Among the most significant recent developments has been the recognition of HER2 as a prognostic factor and target for treatment. In a series of meetings across Europe, 230 experienced oncologists and an expert faculty discussed and voted on how best to treat five women whose cases were chosen to bring out important issues in treatment. In most cases, a range of options were considered appropriate, and intriguing differences emerged between countries in the choices preferred. The following represents a very abbreviated outline of the cases and the management decisions made. Case 1, symptomatic, visceral, metastatic disease overexpressing HER2: most favoured option, trastuzumab plus docetaxel. Case 2, adjuvant chemotherapy in high-risk, hormone-negative, HER2-positive disease: favoured option, FEC-100 for three cycles, followed by three cycles of docetaxel (trastuzumab also given). Case 3, adjuvant endocrine therapy in a postmenopausal woman with hormone-positive, HER2-negative disease: favoured option, tamoxifen for two years followed by aromatase inhibitor (either exemestane or anastrozole). Case 4, inflammatory HER2-positive breast cancer progressing on FEC-100: favoured option, switch to a taxane plus trastuzumab. Case 5, a young woman with hormone-negative, HER2-positive disease who develops symptomatic visceral metastases a year after adjuvant FEC-100 and trastuzumab: favoured option, enrollment in a clinical trial of the HER2 tyrosine kinase inhibitor lapatinib alone or in combination with trastuzumab or chemotherapy. Together, the expression of these preferences and the discussions that followed provide a valuable insight into current practice at a time of exceptionally rapid change in the management of breast cancer.

The effect of neoadjuvant chemotherapy on histologic grade, hormone receptor status, and HER2/neu status in breast carcinoma.

The use of neoadjuvant chemotherapy prior to surgical resection for breast cancer is no longer restricted to patients with locally advanced disease. As preoperative treatment becomes more common, the question arises whether or not such therapy changes important tumor characteristics. The objective of our study is to compare histological grade, hormone receptor status, and HER2/neu expression pre- and post-therapy patients receiving preoperative neo-adjuvant chemotherapy. Forty patients status post-neoadjuvant treatment who had available archived pathologic material pre- and post-therapy were identified. Glass slides were reviewed retrospectively, and tumor grade, hormone receptor status, and HER2/neu expression were compared between the pre- and post-therapy specimens. No significant differences were noted between the pre- and post-specimens for two of the three parameters comprising the modified Bloom-Richardson grade, including degree of tubule formation (p = 0.062) and nuclear pleomorphism (p = 0.086). For mitotic activity, a decrease in score was observed between pre- and post-therapy specimens which was statistically significant (p = 0.021). However, there was no significant difference in the overall modified Bloom-Richardson grade (p = 0.118). Information was available regarding hormone receptor and HER2/neu status in 26 patients (65%). There was no significant difference between pre- and post-treatment specimens for hormone receptor status. However, there were more patients with HER2/neu overexpression after receiving neoadjuvant therapy (p = 0.027). Neoadjuvant therapy resulted in a significant decrease in mitotic count and an increase in the proportion of patients with HER2/neu overexpression. No significant changes were noted for the degree of tubule formation, nuclear pleomorphism, overall Bloom-Richardson score, and hormone receptor status. However, small sample size may be a limitation of these results.

Microenvironment-Mediated Mechanisms of Resistance to HER2 Inhibitors Differ between HER2+ Breast Cancer Subtypes.

Extrinsic signals are implicated in breast cancer resistance to HER2-targeted tyrosine kinase inhibitors (TKIs). To examine how microenvironmental signals influence resistance, we monitored TKI-treated breast cancer cell lines grown on microenvironment microarrays composed of printed extracellular matrix proteins supplemented with soluble proteins. We tested ∼2,500 combinations of 56 soluble and 46 matrix microenvironmental proteins on basal-like HER2+ (HER2E) or luminal-like HER2+ (L-HER2+) cells treated with the TKIs lapatinib or neratinib. In HER2E cells, hepatocyte growth factor, a ligand for MET, induced resistance that could be reversed with crizotinib, an inhibitor of MET. In L-HER2+ cells, neuregulin1-ß1 (NRG1ß), a ligand for HER3, induced resistance that could be reversed with pertuzumab, an inhibitor of HER2-HER3 heterodimerization. The subtype-specific responses were also observed in 3D cultures and murine xenografts. These results, along with bioinformatic pathway analysis and siRNA knockdown experiments, suggest different mechanisms of resistance specific to each HER2+ subtype: MET signaling for HER2E and HER2-HER3 heterodimerization for L-HER2+ cells.

A multidisciplinary approach to the management of breast cancer, part 2: therapeutic considerations.

New approaches to breast cancer treatment have enhanced clinical outcomes and patient care. These approaches include advances in breast irradiation and hormonal and systemic adjuvant therapies. In addition to the identification of new drug targets and targeted therapeutics (eg, trastuzumab), there is renewed re-emphasis in the development of biomarkers for the prediction of response to therapy. One example is the pharmacogenetics of tamoxifen metabolism and the individualization of hormonal therapy. The current treatment of breast cancer continues to evolve rapidly, with new scientific and clinical achievements constantly changing the standard of care and leading to substantial reductions in breast cancer mortality. The goal of this article is to provide clinicians who care for women with breast cancer a multidisciplinary, state-of-the art approach to the treatment of these patients.

Analysis of the pathologic response to primary chemotherapy in patients with locally advanced breast cancer grouped according to estrogen receptor, progesterone receptor, and HER2 status.

PURPOSE: In clinical practice, it is possible to classify breast tumors according to estrogen receptor (ER), progesterone receptor (PgR), and HER2 overexpression: ER negative, PgR negative, and HER2 overexpressing; ER negative, PgR negative, and HER2 negative; ER positive, PgR positive, and HER2 negative; ER positive, PgR positive, and HER2 overexpressing; and the less frequent remaining 4 combinations. The aim of this study was to determine the percentage of pathologic complete response (pCR) in patients with locally advanced breast cancer (LABC) treated with neoadjuvant or primary chemotherapy with anthracyclines and taxanes grouped according to ER, PgR, and HER2 status. PATIENTS AND METHODS: Patients with LABC treated with primary chemotherapy including anthracyclines and taxanes were grouped according to ER, PgR, and HER2 status; pCR rates were analyzed using the chi(2) test; and correlations with a P value of < or = 0.05 were considered statistically significant. RESULTS: A total of 103 patients were treated. Only 100 patients were included for the analysis of pCR. Eighteen patients exhibited pCR. The pCR rate for each subgroup was as follows: 39.1% (9 of 23) had ER-negative, PgR-negative, and HER2-negative disease (P < 0.01); 35.7% (5 of 14) had ER-negative, PgR-negative, and HER2-overexpressing disease; 33.3% (3 of 9) had ER-positive, PgR-positive, and HER2-overexpressing disease; and 2.8% (1 of 36) had ER-positive, PgR-positive, and HER2-negative disease (P < 0.01). CONCLUSION: In patients with LABC, grouping breast tumors according to ER, PgR, and HER2 status can help predict pCR to primary chemotherapy.

Olive oil's bitter principle reverses acquired autoresistance to trastuzumab (Herceptin) in HER2-overexpressing breast cancer cells.

BACKGROUND: A low incidence of breast cancer in the Mediterranean basin suggests that a high consumption of Extra Virgin Olive Oil (EVOO) might confer this benefit. While the anti-HER2 oncogene effects of the main omega-9 fatty acid present in EVOO triacylglycerols (i.e., oleic acid) have been recently described, the anti-breast cancer activities of EVOO non-glyceridic constituents--which consist of at least 30 phenolic compounds--remained to be evaluated. METHODS: Semi-preparative HPLC was used to isolate EVOO polyphenols (i.e., tyrosol, hydroxytyrosol, oleuropein). Both the anti-proliferative and the pro-apoptotic effects of EVOO phenolics were evaluated by using MTT-based quantification of metabolically viable cells and ELISA-based detection of histone-associated DNA fragments, respectively. The nature of the interaction between oleuropein aglycone and the anti-HER2 monoclonal antibody trastuzumab (Herceptin) was mathematically evaluated by the dose-oriented isobologram technique. HER2-specific ELISAs were employed to quantitatively assess both the basal cleavage of the HER2 extracellular domain (ECD) and the expression level of total HER2. The activation status of HER2 was evaluated by immunoblotting procedures using a monoclonal antibody specifically recognizing the tyrosine phosphorylated (Phosphor-Tyr1248) form of HER2. RESULTS: Among EVOO polyphenols tested, oleuropein aglycone was the most potent EVOO phenolic in decreasing breast cancer cell viability. HER2 gene-amplified SKBR3 cells were ~5-times more sensitive to oleuropein aglycone than HER2-negative MCF-7 cells. Retroviral infection of the HER2 oncogene in MCF-7 cells resulted in a "SKBR3-assimilated" phenotype of hypersensitivity to oleuropein aglycone. An up to 50-fold increase in the efficacy of trastuzumab occurred in the presence of oleuropein aglycone. A preclinical model of acquired autoresistance to trastuzumab (SKBR3/Tzb100 cells) completely recovered trastuzumab sensitivity (> 1,000-fold sensitization) when co-cultured in the presence of oleuropein aglycone. Indeed, the nature of the interaction between oleuropein aglycone and trastuzumab was found to be strongly synergistic in Tzb-resistant SKBR3/Tzb100 cells. Mechanistically, oleuropein aglycone treatment significantly reduced HER2 ECD cleavage and subsequent HER2 auto-phosphorylation, while it dramatically enhanced Tzb-induced down-regulation of HER2 expression. CONCLUSION: Olive oil's bitter principle (i.e., oleuropein aglycone) is among the first examples of how selected nutrients from an EVOO-rich "Mediterranean diet" directly regulate HER2-driven breast cancer disease.

Using specific cytotoxics with a targeted mind.

It is largely known that clinical activity of a given cytotoxic agent may vary between different patients. This suggests that breast cancer sub-types can be identified within the endocrine-resistant cohort, each of them with a specific degree of sensitivity to different cytotoxic drugs. Pre-clinical and early clinical data suggest that in the future some molecular markers might have practical value in predicting cytotoxics activity in the clinical setting. The most relevant evidence is summarized below according to the type of cytotoxic agent: (a) Anthracyclines Topoisomerase II alpha (topo II) gene aberrations (amplification or deletion) and/or topo II protein overexpression seem to predict response to topo II inhibitors such as anthracyclines. Of note, HER-2 amplified tumors have a concomitant topo II gene aberration in approximately 50% of cases. Moreover, the majority of hyper-proliferating tumors carry topo II protein overexpression. Early clinical data suggest the existence of a direct correlation between anthracyclines activity and the presence of topo II gene aberration or topo II protein overexpression. (b) Taxanes Microtubule-associated parameters (MTAP) such as the TAU protein, HER-2 gene amplification, and p-53 gene mutations, have been suggested as potential predictive markers for taxanes. Although early clinical data support pre-clinical experiments, the lack of large prospectively designed clinical studies makes it difficult to draw conclusions on the predictive value of these molecular markers. (c) DNA-damaging agents The BRCA 1 protein seems to play a major role in activating DNA repair mechanisms. Loss-of-function BRCA 1 mutations might lead to a substantial deficit in DNA repair mechanisms. This could ultimately translate into increased tumor sensitivity to DNA-damaging agents such as alkylating compounds and platinum-derivates. Pre-clinical and early clinical data seem to suggest that some BRCA 1 gene mutations might render the tumor more sensitive to DNA-damaging agents and clinical studies have recently been activated to investigate properly this hypothesis. A new generation of ongoing clinical studies and a "focused" use of the gene micro-array technology will hopefully clarify the complex interaction existing between molecular targets and cytotoxic drug activity. This "targeted" approach to chemotherapy might ultimately lead to a more effective strategy in breast cancer medical treatment.

Herceptin sensitizes ErbB2-overexpressing cells to apoptosis by reducing antiapoptotic Mcl-1 expression.

PURPOSE: Monoclonal antibodies, such as herceptin and trastuzumab, against the epidermal growth factor receptor ErbB2 (also known as HER2/neu) are an effective therapy for breast cancer patients with overexpression of ErbB2. Herceptin, in combination with standard chemotherapy, such as taxol or etoposide, gives a synergistically apoptotic response in breast tumors. EXPERIMENTAL DESIGN: The mechanism underlying this synergy between chemotherapy and herceptin treatment is not well understood. Herein, we have determined that addition of herceptin, sensitized breast cancer cell lines MDA-MB-231 and MCF-7 to etoposide- or taxol-induced apoptosis. RESULTS: This treatment resulted in reduced expression of ErbB2 and the antiapoptotic Bcl-2 family member Mcl-1 in MDA-MB-231 cells. Using antisense oligonucleotides against Mcl-1, MDA-MB-231 cells were rendered sensitive to etoposide-induced apoptosis similar to herceptin, but combined treatment of antisense against Mcl-1 and herceptin failed to give a significant increase in apoptosis. In 29 human breast tumors immunostained for ErbB2 and Mcl-1, we found that when ErbB2 was overexpressed, there was a corresponding increase in Mcl-1 expression. DISCUSSION: Using murine fibroblasts that express human ErbB2, but no other ErbB family member (NE2), these cells showed resistance to both taxol- and etoposide-induced apoptosis compared with parental cells. In addition, NE2 cells preferentially express the antiapoptotic Bcl-2 family member Mcl-1 compared with parental cells, and treatment with herceptin reduces Mcl-1 expression. Taken together, these results suggest that herceptin sensitizes ErbB2-overexpressing cells to apoptosis by reducing antiapoptotic Mcl-1 protein levels.

Development and therapeutic options for the treatment of raloxifene-stimulated breast cancer in athymic mice.

PURPOSE: Selective estrogen receptor modulators (SERM) are used for the treatment and prevention of breast cancer (tamoxifen) and osteoporosis (raloxifene). Mechanisms of tamoxifen-resistance in breast cancer are incompletely understood but current research is focused on crosstalk between growth factor receptors and the estrogen receptor alpha (ERalpha) pathway. There is increasing clinical use of raloxifene for the treatment of osteoporosis, but the widespread use of this SERM will have consequences for the treatment of breast cancer in raloxifene-exposed women. EXPERIMENTAL DESIGN: We took the strategic step of developing a raloxifene-resistant tumor (MCF-7RALT) model in vivo and investigating the mechanisms responsible for resistance. RESULTS: MCF-7RALT tumors exhibited phase I SERM resistance, growing in response to SERMs and 17beta-estradiol. Epidermal growth factor receptor/HER1 and HER2/neu mRNAs were increased in MCF-7RALT tumors. The HER2/neu blocker, trastuzumab, but not the epidermal growth factor receptor blocker, gefitinib, decreased the growth of MCF-7RALT tumors in vivo. Consequently, trastuzumab decreased prosurvival/proliferative proteins: phospho-HER2/neu, total HER2/neu, phospho-Akt (protein kinase B), glycogen synthetase kinase-3, cyclin D1, and the antiapoptotic protein X chromosome-linked inhibitor of apoptosis, whereas increasing the proapoptotic protein, caspase-7, in raloxifene-treated MCF-7RALT tumors. Interestingly, ERalpha protein was overexpressed in untreated MCF-7RALT tumors and hyperactivated in cells derived from these tumors. Only fulvestrant completely inhibited the growth and ERalpha activity of MCF-7RALT tumors. The coactivator of ERalpha, amplified in breast cancer-1 protein was modestly increased in the raloxifene-treated MCF-7RALT tumors and increased both basal and estradiol-induced activity of ERalpha in cells derived from the MCF-7RALT tumors. CONCLUSIONS: These results suggest that overexpression and increased activity of HER2/neu might be responsible for the development of raloxifene-resistant breast cancer. The results also suggest that increased expression of basal activity of ERalpha could contribute to the hypersensitivity of MCF-7RALT tumors in response to estradiol because only fulvestrant blocked growth and ERalpha activity.