Cancer Research Cell Death Mechanisms and Cancer Therapy Genetics and Biology of Brain Cancer
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
Cancer Research 67, 4190, May 1, 2007. doi: 10.1158/0008-5472.CAN-06-3316
© 2007 American Association for Cancer Research
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplementary Data
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Palmieri, D.
Right arrow Articles by Steeg, P. S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Palmieri, D.
Right arrow Articles by Steeg, P. S.

Cell, Tumor, and Stem Cell Biology

Her-2 Overexpression Increases the Metastatic Outgrowth of Breast Cancer Cells in the Brain

Diane Palmieri1, Julie L. Bronder1, Jeanne M. Herring4, Toshiyuki Yoneda5, Robert J. Weil6, Andreas M. Stark7, Raffael Kurek8, Eleazar Vega-Valle4, Lionel Feigenbaum4, Douglas Halverson1, Alexander O. Vortmeyer2, Seth M. Steinberg3, Kenneth Aldape9 and Patricia S. Steeg1

1 Laboratory of Molecular Pharmacology, Women's Cancers Section, National Cancer Institute; 2 Surgical Neurology Branch, National Institute of Neurological Disorders and Stroke; and 3 Biostatistics and Data Management Section, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, Maryland; 4 Laboratory Animal Sciences Program, Science Applications International Corporation, National Cancer Institute, NIH, Frederick, Maryland; 5 University of Texas Health Science Center at San Antonio, San Antonio, Texas; 6 Brain Tumor Institute, Cleveland Clinic, Cleveland, Ohio; 7 University of Schleswig-Holstein Medical Center, Kiel, Germany; 8 Department of Obstetrics and Gynecology, University of Tuebingen, Tuebingen, Germany; and 9 Department of Neurosurgery, M. D. Anderson Cancer Center, Houston, Texas

Requests for reprints: Diane Palmieri, Laboratory of Molecular Pharmacology, Women's Cancers Section, National Cancer Institute, NIH, Building 37, MSC 4254, Bethesda, MD 20892. Phone: 301-594-0685; Fax: 301-402-8910; E-mail: palmierd{at}mail.nih.gov.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Retrospective studies of breast cancer patients suggest that primary tumor Her-2 overexpression or trastuzumab therapy is associated with a devastating complication: the development of central nervous system (brain) metastases. Herein, we present Her-2 expression trends from resected human brain metastases and data from an experimental brain metastasis assay, both indicative of a functional contribution of Her-2 to brain metastatic colonization. Of 124 archival resected brain metastases from breast cancer patients, 36.2% overexpressed Her-2, indicating an enrichment in the frequency of tumor Her-2 overexpression at this metastatic site. Using quantitative real-time PCR of laser capture microdissected epithelial cells, Her-2 and epidermal growth factor receptor (EGFR) mRNA levels in a cohort of 12 frozen brain metastases were increased up to 5- and 9-fold, respectively, over those of Her-2–amplified primary tumors. Co-overexpression of Her-2 and EGFR was also observed in a subset of brain metastases. We then tested the hypothesis that overexpression of Her-2 increases the colonization of breast cancer cells in the brain in vivo. A subclone of MDA-MB-231 human breast carcinoma cells that selectively metastasizes to brain (231-BR) overexpressed EGFR; 231-BR cells were transfected with low (4- to 8-fold) or high (22- to 28-fold) levels of Her-2. In vivo, in a model of brain metastasis, low or high Her-2–overexpressing 231-BR clones produced comparable numbers of micrometastases in the brain as control transfectants; however, the Her-2 transfectants yielded 3-fold greater large metastases (>50 µm2; P < 0.001). Our data indicate that Her-2 overexpression increases the outgrowth of metastatic tumor cells in the brain in this model system. [Cancer Res 2007;67(9):4190–8]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The Her-2 receptor tyrosine kinase, a member of the Her superfamily, is one of the most studied signal transduction pathways in cancer (reviewed in ref. 1). Amplification or overexpression of Her-2 is detected in ~23% to 26% of primary breast tumors (2) and correlated with poor patient survival (3). Transfection data, genetically engineered mice, and the use of pharmacologic inhibitors revealed effects on tumor cell proliferation, colonization and motility in vitro, and tumorigenicity and metastasis to the lung and bone in vivo (4–11). Trastuzumab (Herceptin), a recombinant humanized monoclonal antibody to Her-2, has shown clinical activity in combination with chemotherapy in the metastatic and adjuvant settings (12–14). Multiple new Her-2–directed therapeutics are in development and clinical testing.

Historically, symptomatic brain metastases develop in 10% to 20% of patients with metastatic breast cancer, most often following systemic disease at other sites, and are associated with a dismal prognosis (reviewed in refs. 15, 16). Retrospective studies of breast cancer patients who developed brain metastases revealed an enrichment for hormone receptor–negative (17–22) and epidermal growth factor receptor (EGFR)–positive (21) primary tumors, lymph node metastases (17, 22), and young patient age (20, 21, 23).

The relationship of Her-2 overexpression in primary breast tumors and the development of brain metastasis has been the subject of considerable investigation. Bendell et al. reported that 34% of breast cancer patients with Her-2–positive tumors developed brain metastases within 16 months of diagnosis of metastatic disease and 6 months of trastuzumab treatment (24). Fifty percent of the patients were responding to therapy or had stable disease at other sites at the time of brain relapse. Fifty percent of patients died of progressive central nervous system (CNS) disease. Similar results were reported by several other groups (17, 21, 25–28). In a study of isolated brain metastases, patients with Her-2–positive tumors, as assessed by fluorescence in situ hybridization (FISH), exhibited a trend (P = 0.09) for development of brain metastases (29). Brain metastatic relapses were also observed in the trastuzumab adjuvant trials, indicating that this problem will not abate with earlier treatment (13). In contrast to these reports, a cohort study by Lai et al. (30) and an autopsy study by Tham et al. (31) found no association between Her-2 expression and brain metastasis. In the present study, we avoided correlative studies using primary tumors and determined the incidence of Her-2 overexpression in a large cohort of surgically resected brain metastases of breast cancer, showing Her-2 enrichment in the metastatic lesions. Furthermore, we show coordinate EGFR/Her-2 overexpression in a subpopulation of brain metastases.

The possible causes of increased brain metastases among Her-2–positive breast cancer patients are multiple: (a) Her-2 overexpression may affect the natural history of breast cancer to accelerate brain progression. (b) Poor penetration of the blood-brain barrier (BBB) by trastuzumab may render the brain a "sanctuary" site for metastases, and/or (c) the increased life span of patients receiving trastuzumab therapy may permit brain metastases to become evident. Resolution of these issues has been hampered by a lack of model systems for studying the biology of brain metastases. We developed a quantitative experimental metastasis assay using a brain trophic clone of human MDA-MB-231 breast carcinoma cells (231-BR). Transfection of Her-2 into 231-BR cells increased brain metastatic colonization 3-fold, providing the first evidence that Her-2 overexpression affects the natural history of breast cancer brain metastatic growth.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Tissue. Samples were coded with no patient identifiers and approved by the National Cancer Institute (NCI) Office of Human Subjects Research. Diagnosis and histopathologic characteristics were confirmed by a single pathologist before use in the study.

Laser capture microdissection, RNA isolation, and amplification. Sections of frozen tissue (8 µm) were stained using a Histogene Laser Capture Microdissection (LCM) Frozen Section Staining kit (Arcturus) following the manufacturer's instructions. For each sample, using the Pix Cell II LCM System (Arcturus), ~2,500 laser-captured pulses were collected in a 15-min period. Captured cells were lysed in PicoPure RNA Extraction buffer (PicoPure RNA Isolation kit, Arcturus) for 30 min at 55°C, and total RNA was extracted. Quality and quantity of RNA was assessed using the RNA 6000 Pico Assay for the 2100 Bioanalyzer (Agilent Technologies). A minimum of 10 ng RNA was then subjected to two rounds of T7 RNA polymerase–catalyzed amplification using the RiboAmp RNA Amplification kit (Arcturus). Quality and quantity of the amplified RNA was assessed using the RNA 6000 Nano Assay (Agilent Technologies). A reference RNA sample consisting of a pool of six breast cancer cell lines (MCF7, ZR-75, BT-474, BT-549, MDA-MB-231, and T-47-D) was simultaneously amplified.

Quantitative real-time PCR. Double-stranded cDNA was prepared using the second-round synthesis protocol from the RiboAmp RNA amplification kit (Arcturus). Real-time quantitative PCR reactions consisted of 1x SybrGreen Supermix (Bio-Rad), 0.3 mmol/L forward and reverse primers, and 10 ng cDNA. Cycling conditions consisted of a three-step amplification and melt curve analysis using the iQ5 Real-time PCR Detection System (Bio-Rad). Annealing temperatures and primer sequences are listed in Supplementary Table S1. For generating a standard curve, amplified cDNA from the reference sample detailed above was used in a 5-fold dilution series of 100 to 0.16 ng cDNA per reaction. Relative gene expression was calculated by dividing the specific expression value (starting quantity, ng) by the glyceraldehyde-3-phosphate dehydrogenase expression value.

Assessment of Her-2 amplification in brain metastatic samples. Archival paraffin-embedded tumor samples were obtained at M.D. Anderson Cancer Center (Houston, TX) from patients with a history of breast cancer who presented with metastases to the CNS parenchyma. Sections from these samples were confirmed to have metastatic breast cancer and subjected to immunohistochemistry for Her-2. Sections were processed and stained as previously described (32) using an anti-Her-2 antibody (Ab-8, Calbiochem) at a 1:500 dilution with microwave antigen retrieval in 10 mmol/L citrate buffer. Scoring was done using a four-tiered system (0, 1+, 2+, and 3+) according to standard clinical practice. Tumors scored as 2+ were subjected to FISH for probes at the Her-2 and CEP17 (centromeric region) loci. FISH was done using the PathVysion Her-2 kit (Vysis) according to the manufacturer's specifications. A ratio > 2.0 was considered positive for Her-2 amplification.

Cell culture. The human MDA-MB-231 BR "brain-seeking" (231-BR) cell line and a companion "bone-seeking" line (231-BO) were previously described (33). The retroviral vector pLEGFP-C1 (BD Biosciences) was transfected into murine fibroblast PT67 packaging cells using Effectene reagent (Qiagen). After 24 h, enhanced green fluorescent protein (EGFP)–expressing cells were selected in the presence of 1 mg/mL G418 (Invitrogen), and colonies were expanded. Virus was harvested and used to infect 231-BR cells for 6 h. The following day, 231-BR cells were selected in the presence of 0.8 mg/mL G418, and EGFP expression in 95% to 99% of the cells was confirmed by fluorescent microscopy. EGFP-expressing cells were then cotransfected with pCMV4.ErbB2 full-length human cDNA (kindly provided by Dr. Dihua Yu, M.D. Anderson Cancer Center) and pSVzeo to confer drug resistance. The sequence of the Her-2 insert in pCMV4.ErbB2 was confirmed by sequencing. Stable colonies were selected in the presence of 0.750 mg/mL zeocin (Invitrogen).

Western blotting. Cells were serum starved overnight and stimulated with 100 ng/mL EGF (Peprotech, Inc.) or 10 ng/mL Heregulin (R&D Systems) for 20 min before lysis. Cells were lysed according to standard procedures, and Western blotting was done. For phosphorylated Her-2 (p-Her-2) and phosphorylated Akt (p-Akt), 100 µg total lysate was used; for all other antibodies, 40 µg total lysate was used. All primary antibodies were obtained from Cell Signaling, unless otherwise indicated. Horseradish peroxidase–conjugated secondary antibodies (Santa Cruz Biotechnology) were used at dilutions of 1:5,000. Proteins were visualized using enhanced chemiluminescence (Cell Signaling) and autoradiography.

Proliferation assays. Cells were plated at a density of 10,000 per well in 96-well plates and incubated for 3 h to allow cells to adhere. The cells were washed with PBS, and media containing either 1% or 10% fetal bovine serum were added to the cells. After 72 h, 0.5 mg/mL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma) was added, and the cells were incubated for 2 h. Following incubation, MTT was dissolved in DMSO, and absorbance was measured at 570 nm. The absorbance recorded on day 3 was divided by the absorbance recorded on day 0 (day of plating), and results are presented as fold growth compared with day 0. Results are representative of three independent experiments, each done in quintuplicate.

Soft agar colonization. Cells (10,000 per well) were plated in 1 mL of culture medium containing 0.3% (w/v) top agar in 24-well plates as described previously (34). After 14 days in culture, colonies (>50 cells) were counted. Results are representative of three independent experiments, each done in triplicate.

Transendothelial migration assays. Human brain microvascular endothelial cells (HBMEC) were purchased from Cell Systems and cultured per manufacturer's guidelines. HBMEC (100,000 cells) were plated on fibronectin-coated Transwell inserts with 3-µm pores (BD Biosciences) and cultured for 5 days to allow a confluent monolayer to form. Medium was replaced daily. On day 5, vector or Her-2–transfected 231-BR cells (25,000 per insert) were added to the top of the insert, and medium containing a chemoattractant was placed in the bottom for 18 h. After incubation, non-migrating cells were removed from the upper surface of the insert. Cells that migrated to the bottom surface were fixed in 4% paraformaldehyde and washed with PBS, and the nuclei were counterstained with Hoechst 33342 (Invitrogen). The membrane was then excised and mounted on a glass slide. Migrating cells were visualized by fluorescent microscope, and the entire bottom surface of the membrane was counted. Data are representative of two experiments conducted using triplicate samples.

Mice and imaging. Animal experiments were conducted under an approved Animal Use Agreement with the NCI. Under isoflurane/O2 anesthesia, twenty 5- to 7-week-old female BALB/c nude mice (Charles River Laboratories) were inoculated with 250,000 cells in 0.1 mL PBS in the left ventricle for each clonal line (see Table 1 ). Mice were euthanized under CO2 asphyxiation after 4 weeks, and brains were excised for imaging.


View this table:
[in this window]
[in a new window]

  		Table 1. In vivo analysis of control and Her-2–overexpressing MDA-MB-231 BR clones

 
EGFP was detected in whole brains by the Maestro 420 In vivo Spectral Imaging System (Cambridge Research and Instrumentation) using software (Nuance Technology) to distinguish or unmix images of fluorescence from multiple sources. After imaging, brains were bisected along the sagittal plane, and the left hemisphere was immediately frozen in ornithine carbamyl transferase compound (for histology). The right hemisphere was fixed in 4% paraformaldehyde for 24 to 48 h at 4°C and then transferred to 20% sucrose overnight at 4°C and frozen (for EGFP detection). Brain sections (10-µm thick) were serially cut and processed for either EGFP microscopy or histology. To detect EGFP, slides were dried at room temperature, and 25 µL Vectashield HardSet Mounting Medium with 4',6-diamidino-2-phenylindole was added (Vector Laboratories). Images were captured on a Zeiss Axioskop 2 microscope using OpenLab software (Improvision). For staining with H&E, a standard protocol was followed.

Ten serial sections every 300 µm through the brain were analyzed using a 5x objective on a Zeiss microscope, containing an ocular grid with squares of 0.8 mm2. Every micrometastasis or large (>50 µm2) metastasis in each section was tabulated; counting was terminated at 300 metastases per section. Data are representative of two experiments conducted. All analysis was carried out by two investigators who were blinded to the various groups.

Statistical analysis. ANOVA with post hoc Dunnett's multiple comparison test was used to assess in vitro functions of vector and Her-2–overexpressing clones for proliferation and colonization experiments. Student's t test was used for transendoethelial cell migration experiments, which were conducted in vector and low Her-2 and vector and high Her-2 clone paired experiments, respectively. All P values are two tailed. All tests for in vitro experiments were done using GraphPad InStat version 3.0 software. For analysis of the Her-2 staining, an exact binomial test was used to compare the observed results with the theoretical (historic) percentage of Her-2 overexpression in primary breast tumors. For in vivo experiments, micrometastases and large metastases were evaluated as the primary end points. A general linear model–based ANOVA that adjusted for the effect of clones nested within each group was used to compare vector versus low Her-2 groups and vector versus high Her-2 groups. All P values are two tailed.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Frequent Her-2 overexpression in brain metastases. To determine the incidence of Her-2 overexpression in brain metastases of breast cancer, formalin-fixed, paraffin-embedded sections of resected brain metastases from patients with documented primary breast cancer were analyzed for Her-2 expression using immunohistochemistry. The median age of the cohort was 51 years, with a range from 27 to 75 years; 33 of the 124 cases were estrogen receptor (ER) positive (26.6%). Immunohistochemical scoring by a neuropathologist was based on a 0 to 1+ versus 2 to 3+ intensity point scale (Fig. 1 ). For the borderline positive (2+) metastases, Her-2 amplification was confirmed by FISH (Fig. 1C and D, inset). Of 124 brain metastases, 45 (36.2%) were Her-2 positive. This is higher than a historical percentage (23–26%) for primary breast tumors (2), statistically significant by the exact binomial test (P = 0.015). Estimates of Her-2 overexpression in primary tumors from the metastatic subset of breast cancer patients are rarely reported, but rates of 18% to 30% have been published from small cohort studies using variable detection methodologies (21, 35–37), also lower than the data reported herein.


Figure 1
View larger version (28K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 1. Immunohistochemical and FISH analyses of Her-2 overexpression in a cohort of 124 brain metastases from breast cancer patients. Archival paraffin-embedded tumor samples were subjected to immunohistochemistry for Her-2 using an anti–Her-2 antibody. Scoring was done using a four-tiered system (0, 1+, 2+, and 3+) according to standard practice. Tumors scored as 0 (A) or 1+ (B) were considered negative for Her-2 overexpression. Tumors scored as 3+ (E) were considered positive for Her-2 overexpression. Tumors scored as 2+ (C and D) were subject to FISH for probes at the Her-2 locus and also CEP17 (centromeric region; inset). A ratio > 2.0 was considered positive for Her-2 amplification. Forty-five of 124 (36.2%) samples were positive for Her-2 overexpression. The comparison of the observed proportion to the historic proportion (2) of primary breast tumors yielded P = 0.015 by exact binomial test.

 
Her superfamily mRNA expression levels. Her-2 heterodimerizes with other Her superfamily receptors to initiate signal transduction. The relative expression of the Her superfamily of receptors was determined by quantitative real-time PCR of laser capture microdissected epithelial cells from 12 frozen resected brain metastases and an equal number of unlinked primary breast tumors (Fig. 2 ). Patient and tumor histopathology data for these samples are listed on Supplementary Table S2 and show a general correspondence of patient age, tumor ER status, and tumor-node-metastasis stage.


Figure 2
View larger version (15K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 2. Expression of Her superfamily receptors in unlinked brain metastases of breast cancer and primary breast tumors. Epithelial cells were laser capture microdissected from 12 resected brain metastases and an equal number of infiltrating ductal carcinomas. Real-time quantitative PCR was performed on amplified cDNA. Data are expressed as relative expression in ratio to glyceraldehyde-3-phosphate dehydrogenase levels for each tumor. +, Her-2–amplified tumors as assessed by FISH.

 
For Her-2, FISH analysis indicated amplification in 25% (3 of 12) of the primary tumors and 42% (5 of 12) of the brain metastases. The FISH-positive primary tumors expressed increased Her-2 mRNA levels compared with unamplified tumors; however, brain metastasis Her-2 expression levels were increased up to 5-fold over that of any of the primary tumors.

Similar trends were observed for EGFR (Her-1). Variability in primary tumor EGFR mRNA levels was observed. Five brain metastases (42%) expressed higher EGFR mRNA levels than any of the primary tumors; levels of EGFR expression were up to 9-fold higher than any of the primary tumors. Coordinate high expression of Her-2 and EGFR mRNAs was observed in 17% (2 of 12) of the brain metastases.

In contrast, Her-3 mRNA expression levels were variable and were not different between primary tumors and brain metastases. One of 12 tumors (8%) coordinately expressed high Her-2 and Her-3. With the exception of one brain metastasis, Her-4 mRNA levels were higher in primary tumors than in brain metastases. Although based on a limited cohort, the data suggest a selection for high Her-2 and EGFR mRNA expression in brain metastases. Of the five Her-2–positive (FISH amplified) brain metastases, coordinate high expression of another superfamily member was observed in 60% (3 of 5).

Transfection of Her-2 into a brain-seeking breast cancer cell line augments anchorage-independent colonization. Although an increased incidence of brain metastasis has been correlated with primary tumor Her-2 overexpression and trastuzumab treatment, the lack of breast cancer brain metastasis model systems has hampered our ability to test the functional contribution of Her-2. Two tissue-specific sublines, a brain-seeking (231-BR) and a bone-seeking (231-BO) subline of human MDA-MB-231 breast carcinoma cells, were isolated by successive rounds of in vivo selection and ex vivo culture (33). Analyses of these lines for several key signaling proteins were assessed under normal growth conditions (Fig. 3A ). Acquisition of brain but not bone metastatic activity was accompanied by increased total and p-EGFR expression. Expression of the Her-2, Her-3, and Her-4 proteins was comparable between the lines and at the lower limit of detection (data not shown). Overexpression of EGFR in the 231-BR cell line was accompanied by increased expression of p-Akt; equivalent levels of total and phosphorylated extracellular signal-regulated kinase (p-Erk) were observed in the three lines. Of the many proposed downstream effectors of aggressiveness, we observed elevated p21 levels in both the bone- and brain-selected lines.


Figure 3
View larger version (26K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 3. Western blot analysis of the MDA-MB-231BR brain-seeking cell line and clonal transfectants. Western blots of total cell lysates. A, acquisition of brain metastatic activity was associated with EGFR overexpression. MDA-MB-231 parental cells (P), and sublines selected for brain-seeking (BR) and bone-seeking (BO) in vivo metastatic patterns were previously described (33). B, Her-2 overexpression alters downstream signaling pathways in 231-BR brain metastatic breast carcinoma cells. 231-BR human breast carcinoma cells stably transfected to express a control vector (vector-1 and vector-2), low levels of Her-2 (low-1 and low-2), or high levels of Her-2 (high-1 and high-2) were serum starved for 16 h and then stimulated with 100 ng/mL EGF for 10 min or 10 ng/mL heregulin for 20 min, targeting Her-2 heterodimerization with EGFR and Her-3, respectively. {alpha}-Tubulin expression was used as a loading control. S, serum starved; E, serum starved then stimulated with EGF; H, serum starved then stimulated with HRG.

 
To develop a model system to examine the relevance of Her-2 overexpression in brain metastases, 231-BR cells were first transduced with EGFP and subsequently transfected with either Her-2 cDNA or a control vector. Given the heterogeneity of Her-2 expression levels among brain metastases (Fig. 2), two low and two high Her-2–expressing clones were selected for characterization compared with two vector control clones. To determine whether the overexpressed Her-2 was functional in signal transduction, clones were serum starved and treated with 100 ng/mL EGF or 10 ng/mL heregulin (HRG), which target Her-2 heterodimerization with either EGFR or Her-3, respectively. Her-2 was overexpressed ~8- and 4-fold (low 1 and low 2, respectively) in the low expressing clones and ~28- and 22-fold (high 1 and high 2, respectively) in the high expressing clones, respectively (Fig. 3B). Her-2 phosphorylation was stimulated by EGF in the low and high Her-2 transfectants and by HRG in the high expressing clones. Expression and ligand-mediated activation of EGFR was observed in all transfectants, consistent with data from the 231-BR line (Fig. 3A). Of the proteins reported to lie downstream of Her-2 signaling, p-Akt was activated by EGF preferentially in the high Her-2–expressing clones, whereas p21 elevation was observed in all Her-2 transfectants; no differences in p-Erk were observed. The data indicate that the overexpressed Her-2 protein was active in signal transduction, with EGFR as a leading heterodimerization partner.

We sought to identify the in vitro biological correlates of Her-2 overexpression on metastatic progression (Fig. 4 ). In 3-day proliferation assays, Her-2–overexpressing clones exhibited a minor (1.4-fold) proliferative advantage compared with controls in low (1.0%) serum (P < 0.0001, ANOVA) but no difference at higher (10%) serum levels (Fig. 4A). Anchorage-independent colonization of Her-2 transfectants was statistically significantly elevated over vector transfectants using low (2%) or high (10%) serum levels (P < 0.0001 for both, ANOVA; Fig. 4B). Motility of the vector and Her-2 transfectants was comparable in response to serum-free media supplemented with EGF or HRG, conditioned media from brain microvascular endothelial cells, or conditioned media supplemented with EGF (data not shown). To mimic human tumor cells migrating through the BBB, we used HBMEC in an assay that replicates aspects of BBB traversal in vitro by measuring the ability of tumor cells to invade through a monolayer of HBMEC. EGF-induced transendothelial cell migration was increased 2-fold (P = 0.0025 and P = 0.04, respectively, t test for paired samples) in high Her-2–expressing cells (Fig. 4C). Transendothelial migration was not statistically significantly increased in the low Her-2–expressing clones, or in any clone in low (1%) serum conditions. The data indicate anchorage-independent colonization as the prime phenotypic effect of Her-2 overexpression in this brain-seeking cell line.


Figure 4
View larger version (25K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 4. In vitro phenotypes of Her-2 overexpression in 231-BR cells. A, proliferation of control and low and high Her-2–expressing transfectants over 3 d using the MTT microtiter plate assay. P < 0.0001 by ANOVA for 1% fetal bovine serum (FBS) with *, P < 0.01 for Dunnett multiple comparison test. B, soft agar colonization in 2% and 10% fetal bovine serum. Colonies were counted after 14 d in culture. P < 0.0001 by ANOVA for 2% and 10% fetal bovine serum with *, P < 0.01 for Dunnett multiple comparison test. C, transendothelial cell migration of control and low and high Her-2–expressing 231-BR cells toward EGF. HMBEC were plated on fibronectin-coated Transwell inserts and cultured for 5 d to establish a monolayer with characteristics of a BBB. EGFP-transduced 231BR cells were added to the top of the insert on day 5 and allowed to migrate for 18 h. GFP-positive tumor cells were counted on the entire bottom surface of the insert. Data are expressed as fold increase in transendothelial migration (TM) compared with vector controls. *, P = 0.0025; **, P = 0.04 by Student's t test for vector and high Her-2–expressing clones, respectively, in paired experiments. #, P = 0.03 by Student's t test for vector and low Her-2–expressing clones, respectively, in paired experiments.

 
Her-2 overexpression increases brain colonization in vivo. To determine whether Her-2 overexpression is associated with the increased formation of breast cancer brain metastases in vivo, vector and low and high Her-2–overexpressing transfectants were injected into the left cardiac ventricle of BALB/c nude mice. The number and size of brain metastases that formed were analyzed after 4 weeks. At necropsy, whole brains were removed, and EGFP imaging was done to identify mice with brain metastases (Fig. 5A ). Brains of mice that received diluent only showed nonspecific autofluorescent scatter at either end of the brain, whereas brains of mice that received 231-BR cells showed foci throughout the brain. Although a general trend of increased fluorescent metastases with Her-2 overexpression was noted in the whole-brain imaging, quantification was difficult because of frequent "carpeting" of the brain by overlapping lesions.


Figure 5
View larger version (71K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 5. 231-BR in vivo model system for quantification of brain metastases. EGFP-labeled 231-BR brain-seeking cells were injected into the left cardiac ventricle of BALB/c nude mice, and brain metastases were determined 4 wks later. A, ex vivo whole-brain images were captured using the Maestro 420 In vivo Spectral Imaging System. Representative images from each group. Negative control is a representative image from a mouse that did not receive an injection of tumor cells. Autofluorescence is routinely noted on the periphery of the tissue. B, histologic images representative of micrometastases and large brain metastases. Left, EGFP fluorescence; middle, 4',6-diamidino-2-phenylindole (DAPI)–stained nuclei; right, H&E-stained images in a successive section. Magnification, x200.

 
After imaging, the brain was bisected, and 10 sagittal sections were prepared at 300-µm intervals through one hemisphere. The sections were processed for either EGFP visualization or H&E stained for histology. The presence of brain metastases, which was confirmed by a neuropathologist, occurred in both the parenchyma and leptomeninges. Most of the metastases in each section could be classified as micrometastases, comprising few tumor cells (Fig. 5B, top). For the control transfectants, micrometastases represented 98% of the lesions identified. Variable numbers of micrometastases were formed by the clonal control transfectants, which may reflect heterogeneity in tumor cell survival in this site. To distinguish micrometastases from larger metastases that are diagnosed clinically, each lesion was viewed through an ocular micrometer, and those with an area > 50 µm2 were designated as "large" (Fig. 5B, bottom). This is similar to the size of a clinically detectable metastasis in the human brain by magnetic resonance imaging.

A statistically significant difference was noted in the number of large metastatic lesions formed by the Her-2–overexpressing 231-BR cells (Table 1). The number of total metastases did not vary between vector control and Her-2–overexpressing clones, indicating that the tumor cells arrested, extravasated into the brain, and formed micrometastases with equal efficiency. However, both the low and high Her-2–overexpressing clones formed 3-fold greater large metastases compared with the vector transfectants (P < 0.0001 for both), a trend consistent with the high rate of clinical detection of brain metastases in patients with Her-2–amplified tumors.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Brain metastases of breast and other cancers seem to be increasing in incidence. For breast cancer patients with Her-2–positive tumors, metastases to the brain are also reportedly occurring in patients responding to treatment or with stable disease at other sites. Given the dire cognitive and physical consequences of the development of brain metastases and their treatment, reflected in the dismal 1-year patient survival rate (20%; ref. 15), it is important that we obtain a molecular understanding of metastasis to this site. The data reported herein support three conclusions: (a) Her-2 overexpression is enriched in brain metastases of breast cancer, confirming Her-2 as a molecular target for metastasis to this site. (b) Coordinate high expression of Her-2 and EGFR occurs in a subset of brain metastases. (c) Overexpression of Her-2 elevated the number of large brain metastases 3-fold in an EGFR-overexpressing 231-BR experimental metastasis model system, providing the first evidence that Her-2 can affect the natural history of breast cancer metastasis to the brain.

We determined Her-2 overexpression in a large cohort of resected brain metastases of breast cancer. With the classification for Her-2 positivity defined as 3+ immunohistochemical staining or 2+ immunohistochemical staining and gene amplification by FISH, 36.2% of resected brain metastases of breast cancer were Her-2 positive. A comparable technical approach to defining Her-2 positivity was used for entry into trastuzumab clinical trials (13). This increase in percentage of Her-2 overexpression represents a 42% to 61% enrichment over reported primary tumor Her-2 positivity (23–26%; ref. 2) and supports a predilection of Her-2–positive tumors for brain metastasis. These data stand in agreement with observational studies reporting a correlation of primary tumor Her-2 positivity, or trastuzumab treatment with the development of brain metastases (21, 24–28). Where such correlations were not observed, several explanations may be germane. For instance, methods of detecting Her-2 overexpression vary among studies. Protein-based methods, such as Western blotting, can add dilutional artifacts and have been reported to be inferior to FISH (2).

Not only is the incidence of Her-2 positivity enriched in brain metastases, but based on a limited cohort of unlinked primary tumors and brain metastases, Her-2 mRNA expression levels may be higher than in primary tumors. LCM of epithelial cells from each tumor permitted quantification of Her-2 mRNA levels without dilution artifacts associated with varying numbers of stromal cells. Each of the five Her-2 FISH-amplified brain metastases had higher Her-2 mRNA levels than the FISH-amplified primary tumors (i.e., up to 5-fold higher). A similar trend was observed for EGFR. Increased EGFR expression was also found in the brain-seeking but not the bone-seeking sublines of our MDA-MB-231 model system, suggesting its potential importance. No trend was observed for Her-3 mRNA levels, providing evidence of specificity in expression patterns. Taken together, the data suggest a selection for high EGFR and Her-2 expression in brain metastases that merits further study in larger cohorts.

Combined high EGFR and Her-2 expression was observed in two of five Her-2–positive brain metastases. Coordinate overexpression of Her-2 and EGFR is seldom observed in breast cell lines (38). In tumor cohorts, coordinate overexpression measured by immunohistochemistry to total or phosphorylated receptors, or gene amplification, was found in 2% to 13% of all breast carcinomas (39, 40) and 13% of Her-2–positive tumors (41). Where measured, combined overexpression correlated with poor disease-free survival (40, 41). Grupka et al. (42) used immunohistochemistry to determine Her-2 and EGFR status in a limited brain metastatic cohort and reported that 18% of the lesions were coordinately positive. These data support our mRNA coexpression conclusions at the protein level. Zhan et al. (43) recently reported that EGFR/Her-2 heterodimers in MCF10A cells, compared with Her-2 homodimers, preferentially activated the phosphatidylinositol 3-kinase and phospholipase c{gamma}1 pathways and induced invasion in three-dimensional cultures. These data suggest that coordinate expression of EGFR and Her-2 may endow breast tumors with added invasive properties. The data suggest that the coordinate EGFR/Her-2–positive overexpressing tumors may represent an interesting subset. This subset of breast tumors with Her-2/EGFR overexpression is represented by our MDA-MB-231-BR Her-2 transfectants, which exhibit both high EGFR and Her-2 expression.

An in vivo model system to quantify brain metastases was developed using intracardiac injection of 231-BR cells. Initial experiments confirmed that brain metastatic potential directly associated with the number of tumor cells injected and the time after injection. Although imaging is convenient, a careful cross-comparison with histology indicated the latter as a superior method to quantify brain metastases. In our experience, an imageable brain metastasis could be a single large metastasis or could result from a closely spaced collection of micrometastases. A histologic approach was therefore employed for quantification of our in vivo data and was also used in a similar model (44).

Transfection of Her-2 into 231-BR cells resulted in a 3-fold increase in large (>50 µm2) brain metastases, providing the first evidence that Her-2 affects the natural history of breast cancer progression to the brain. Surprisingly, both low (4- to 8-fold) and high (22- to 28-fold) Her-2–overexpressing clones exhibited increased brain colonization. Many of the large brain metastases observed in the Her-2 transfectants were parenchymal (data not shown).

In other reported model systems, Her-2 transfection increased metastasis to the lung or bones (5, 7, 8, 45–47). The brain may not constitute an obvious extension of these trends. Brain tissue is protected by a BBB consisting of endothelial cells linked by continuous tight junctions, pericytes, a basement membrane, and the feet of astrocytes. Intravasation of this complex barrier cannot be assumed to occur at rates equivalent to arrest by size restriction in a lung capillary. The brain microenvironment is poorly described but seems distinct from that of other organs and may differentially influence the outgrowth of metastases (15). One of two of these theoretical factors seemed to be important in our model system. Her-2–positive and Her-2–negative tumor cells intravasated into the brain and formed micrometastases at comparable frequencies; the outgrowth of Her-2–positive tumor cells to form large metastases in the brain microenvironment was augmented. These in vivo data closely compare with the in vitro analysis; colonization in soft agar was the only assay in which Her-2 transfectants outperformed the vector controls under all conditions tested.

A host of signaling pathways may mediate Her-2 aggressiveness. Both Her-2 phosphorylation and increased p21 expression were associated with increased brain metastatic potential in this system. p21Waf1/Cip1 was classically described as an inhibitor of cell cycle progression but has also been linked to both innate tumor cell aggressiveness and chemotherapeutic resistance. In a breast cancer cohort, phosphorylation or cytoplasmic localization of p21 was associated with Her-2 overexpression (48); the subcellular distribution and influence of p21 on tumor cell sensitivity to chemotherapeutic compounds was modulated by the Her-2 pathway (49). The role of p21 in this model system, as well as other potential signaling mechanisms, remains under study. It will be of interest to determine the role of Her-2 overexpression in brain metastasis in the absence of tumor cell EGFR overexpression and to compare the signaling pathways involved. Given the causal relationship of Her-2 overexpression and brain colonization showed herein, in vitro and in vivo, the efficacy of small BBB-permeable inhibitors of Her-2 will be of great interest.


   Acknowledgments
 
Grant support: Intramural Research Program of the National Cancer Institute, Center for Cancer Research, NIH.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   Footnotes
 
Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).

D. Palmieri and J.L. Bronder contributed equally to this work.

Current address for R. Kurek: Merck KGaA, Frankfurter Str. 250, D 64293 Darmstadt, Germany.

Received 9/ 8/06. Revised 2/ 5/07. Accepted 2/16/07.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Hynes NE, Lane HA. ERBB receptors and cancer: the complexity of targeted inhibitors. Nat Rev Cancer 2005;5:341–4.[CrossRef][Medline]
  2. Press MF, Sauter G, Bernstein L, et al. Diagnostic evaluation of HER-2 as a molecular target: an assessment of accuracy and reproducibility of laboratory testing in large, prospective, randomized clinical trials. Clin Cancer Res 2005;11:6598–607.[Abstract/Free Full Text]
  3. Slamon DJ, Godolphin W, Jones LA, et al. Studies of the HER-2/neu proto-oncogene in human breast and ovarian cancer. Science 1989;244:707–12.[Abstract/Free Full Text]
  4. Liu Y, el-Ashry D, Chen D, Ding IY, Kern FG. MCF-7 breast cancer cells overexpressing transfected c-erbB-2 have an in vitro growth advantage in estrogen-depleted conditions and reduced estrogen-dependence and tamoxifen-sensitivity in vivo. Breast Cancer Res Treat 1995;34:97–117.[CrossRef][Medline]
  5. Tan M, Yao J, Yu D. Overexpression of the c-erb-B-2 gene enhanced intrinsic metastatic potential in human breast cancer cells without increasing their transformation abilities. Cancer Res 1997;57:1199–205.[Abstract/Free Full Text]
  6. Khalili P, Arakelian A, Chen G, Singh G, Rabbani SA. Effect of Herceptin on the development and progression of skeletal metastases in a xenograft model of human breast cancer. Oncogene 2005;24:6657–66.[CrossRef][Medline]
  7. Guy C, Webster M, Schaller M, Parsons T, Cardiff R, Muller W. Expression of the neu protooncogene in the mammary epithelium of transgenic mice induces metastatic disease. Proc Natl Acad Sci U S A 1993;89:10578–82.
  8. Moody SE, Sarkisian CJ, Hahn KT, et al. Conditional activation of Neu in the mammary epithelium of transgenic mice results in reversible pulmonary metastasis. Cancer Cell 2002;2:451–61.[CrossRef][Medline]
  9. Marone R, Hess D, Dankort D, Muller WJ, Hynes NE, Badache A. Memo mediates ErbB2-driven cell motility. Nat Cell Biol 2004;6:515–22.[CrossRef][Medline]
  10. Faltus T, Yuan J, Zimmer B, et al. Silencing of the HER2/neu gene by siRNA inhibits proliferation and induces apoptosis in HER2/neu-overexpressing breast cancer cells. Neoplasia 2004;6:786–95.[CrossRef][Medline]
  11. Pietras RJ, Pegram MD, Finn RS, Maneval DA, Slamon DJ. Remission of human breast cancer xenografts on therapy with humanized monoclonal antibody to HER-2 receptor and DNA-reactive drugs. Oncogene 1998;17:2235–49.[CrossRef][Medline]
  12. Slamon D, Leyland-Jones B, Shak S, et al. Use of chemotherapy plus a monoclonal antibody against Her2 for metastatic breast cancer that overexpresses Her2. N Engl J Med 2001;344:783–92.[Abstract/Free Full Text]
  13. Piccart-Gebhart MJ, Procter M, Leyland-Jones B, et al. Trastuzumab after adjuvant chemotherapy in HER2-positive breast cancer. N Engl J Med 2005;353:1659–72.[Abstract/Free Full Text]
  14. Romond EH, Perez EA, Bryant J, et al. Trastuzumab plus adjuvant chemotherapy for operable HER2-positive breast cancer. N Engl J Med 2005;353:1673–84.[Abstract/Free Full Text]
  15. Weil R, Palmieri D, Bronder J, Stark A, Steeg P. Breast cancer metastasis to the central nervous system. Am J Pathol 2005;167:913–20.[Abstract/Free Full Text]
  16. Lin N, Bellon J, Winer E. CNS metastases in breast cancer. J Clin Oncol 2004;22:3608–17.[Abstract/Free Full Text]
  17. Pestalozzi BC, Zahrieh D, Price KN, et al. Identifying breast cancer patients at risk for central nervous system (CNS) metastases in trials of the International Breast Cancer Study Group (IBCSG). Ann Oncol 2006;17:935–44.[Abstract/Free Full Text]
  18. Clark G, Sledge GW, Osborne C, McGuire W. Survival from first recurrence: relative importance of prognostic factors in 1,015 breast cancer patients. J Clin Oncol 1987;5:55–61.[Abstract]
  19. Slimane K, Andre F, Delaloge S, et al. Risk factors for brain relapse in patients with metastatic breast cancer. Ann Oncol 2004;15:1640–4.[Abstract/Free Full Text]
  20. Evans A, James J, Cornford E, et al. Brain metastases from breast cancer: identification of a high-risk group. Clin Oncol 2004;16:345–9.[CrossRef]
  21. Hicks D, Short S, Prescott N, et al. Breast cancers with brain metastases are more likely to be estrogen receptor negative, express the basal cytokeratin CK5/6 and over-express Her-2 or EGFR. Am J Surg Path 2006;30:1097–104.[Medline]
  22. Ryberg M, Nielsen D, Osterlind K, Andersen PK, Skovsgaard T, Dombernowsky P. Predictors of central nervous system metastasis in patients with metastatic breast cancer. A competing risk analysis of 579 patients treated with epirubicin-based chemotherapy. Breast Cancer Res Treat 2005;91:217–25.[CrossRef][Medline]
  23. Carey LA, Ewend MG, Metzger R, et al. Central nervous system metastases in women after multimodality therapy for high risk breast cancer. Breast Cancer Res Treat 2004;88:273–80.[CrossRef][Medline]
  24. Bendell J, Domchek S, Burstein H, et al. Central nervous system metastases in women who receive trastuzumab-based therapy for metastatic breast carcinoma. Cancer 2003;97:2972–7.[CrossRef][Medline]
  25. Shmueli E, Wigler N, Inbar M. Central nervous system progression among patients with metastatic breast cancer responding to trastuzumab treatment. Eur J Cancer 2004;40:379–82.[CrossRef][Medline]
  26. Lower E, Blau R, Bismayer J, et al. Increased brain metastasis detected in metastatic breast cancer patients receiving Herceptin [Abstract 351]. Breast Cancer Res Treat 2001;69:271.
  27. Clayton A, Danson S, Jolly S, et al. Incidence of cerebral metastases in patients treated with trastuzumab for metastatic breast cancer. Br J Cancer 2004;91:639–43.[Medline]
  28. Stemmler HJ, Kahlert S, Siekiera W, Untch M, Heinrich B, Heinemann V. Characteristics of patients with brain metastases receiving trastuzumab for HER2 overexpressing metastatic breast cancer. Breast 2006;15:219–25.[CrossRef][Medline]
  29. Burstein HJ, Lieberman G, Slamon DJ, Winer EP, Klein P. Isolated central nervous system metastases in patients with HER2-overexpressing advanced breast cancer treated with first-line trastuzumab-based therapy. Ann Oncol 2005;16:1772–7.[Abstract/Free Full Text]
  30. Lai R, Dang C, Malkin M, Abrey L. The risk of central nervous system metastases after trastuzumab therapy in patients with breast carcinoma. Cancer 2004;101:810–6.[CrossRef][Medline]
  31. Tham YL, Sexton K, Kramer R, Hilsenbeck S, Elledge R. Primary breast cancer phenotypes associated with propensity for central nervous system metastases. Cancer 2006;107:696–704.[CrossRef][Medline]
  32. Simmons ML, Lamborn KR, Takahashi M, et al. Analysis of complex relationships between age, p53, epidermal growth factor receptor, and survival in glioblastoma patients. Cancer Res 2001;61:1122–8.[Abstract/Free Full Text]
  33. Yoneda T, Williams P, Hiraga T, Niewolna M, Nishimura R. A bone seeking clone exhibits different biological properties from the MDA-MB-231 parental human breast cancer cells and a brain-seeking clone in vivo and in vitro. J Bone Miner Res 2001;16:1486–95.[CrossRef][Medline]
  34. Leone A, Flatow U, VanHoutte K, Steeg PS. Transfection of human nm23-H1 into the human MDA-MB-435 breast carcinoma cell line: effects on tumor metastatic potential, colonization, and enzymatic activity. Oncogene 1993;8:2325–33.[Medline]
  35. Miller K, Weathers T, Hanley L, et al. Occult central nervous system involvement in patients with metastatic breast cancer: prevalence, predictive factors and impact on overall survival. Ann Oncol 2003;14:1072–7.[Abstract/Free Full Text]
  36. Harris LN, Liotcheva V, Broadwater G, et al. Comparison of methods of measuring HER-2 in metastatic breast cancer patients treated with high-dose chemotherapy. J Clin Oncol 2001;19:1698–706.[Abstract/Free Full Text]
  37. Emi Y, Kitamura K, Shikada Y, Kakeji Y, Takahashi I, Tsutsui S. Metastatic breast cancer with HER2/neu-positive cells tends to have a morbid prognosis. Surgery 2001;131:S217–21.
  38. Konecny GE, Pegram MD, Venkatesan N, et al. Activity of the dual kinase inhibitor lapatinib (GW572016) against HER-2-overexpressing and trastuzumab-treated breast cancer cells. Cancer Res 2006;66:1630–9.[Abstract/Free Full Text]
  39. Bhargava R, Gerald WL, Li AR, et al. EGFR gene amplification in breast cancer: correlation with epidermal growth factor receptor mRNA and protein expression and HER-2 status and absence of EGFR-activating mutations. Mod Pathol 2005;18:1027–33.[CrossRef][Medline]
  40. DiGiovanna MP, Stern DF, Edgerton SM, Whalen SG, Moore II D, Thor AD. Relationship of epidermal growth factor receptor expression to ErbB-2 signaling activity and prognosis in breast cancer patients. J Clin Oncol 2005;23:1152–60.[Abstract/Free Full Text]
  41. Hudelist G, Kostler WJ, Czerwenka K, et al. Her-2/neu and EGFR tyrosine kinase activation predict the efficacy of trastuzumab-based therapy in patients with metastatic breast cancer. Int J Cancer 2006;118:1126–34.[CrossRef][Medline]
  42. Grupka N, Lear-Kaul K, Kleinschmidt-DeMasters B, Singh M. Epidermal growth factor receptor status in breast cancer metastases to the central nervous system. Arch Pathol Lab Med 2004;128:974–9.[Medline]
  43. Zhan L, Xiang B, Muthuswamy SK. Controlled activation of ErbB1/ErbB2 heterodimers promote invasion of three-dimensional organized epithelia in an ErbB1-dependent manner: implications for progression of ErbB2-overexpressing tumors. Cancer Res 2006;66:5201–8.[Abstract/Free Full Text]
  44. Kim L, Huang S, Lu W, Lev DC, Price J. Vascular endothelial growth factor expression promotes the growth of breast cancer brain metastases in nude mice. Clin Exp Metastasis 2004;21:107–18.[CrossRef][Medline]
  45. Klos KS, Wyszomierski SL, Sun M, et al. ErbB2 increases vascular endothelial growth factor protein synthesis via activation of mammalian target of rapamycin/p70S6K leading to increased angiogenesis and spontaneous metastasis of human breast cancer cells. Cancer Res 2006;66:2028–37.[Abstract/Free Full Text]
  46. Li Y, Pan Y, Wei Y, et al. Upregulation of CXCR4 is essential for HER2-mediated tumor metastasis. Cancer Cell 2004;6:459–69.[CrossRef][Medline]
  47. Tsai MS, Shamon-Taylor LA, Mehmi I, Tang CK, Lupu R. Blockage of heregulin expression inhibits tumorigenicity and metastasis of breast cancer. Oncogene 2003;22:761–8.[CrossRef][Medline]
  48. Xia W, Chen JS, Zhou X, et al. Phosphorylation/cytoplasmic localization of p21Cip1/WAF1 is associated with HER2/neu overexpression and provides a novel combination predictor for poor prognosis in breast cancer patients. Clin Cancer Res 2004;10:3815–24.[Abstract/Free Full Text]
  49. Menendez JA, Mehmi I, Lupu R. Heregulin-triggered Her-2/neu signaling enhances nuclear accumulation of p21WAF1/CIP1 and protects breast cancer cells from cisplatin-induced genotoxic damage. Int J Oncol 2005;26:649–59.[Medline]



This article has been cited by other articles:


Home page
 Clin. Cancer Res.Home page
D. Palmieri, P. R. Lockman, F. C. Thomas, E. Hua, J. Herring, E. Hargrave, M. Johnson, N. Flores, Y. Qian, E. Vega-Valle, et al.
Vorinostat Inhibits Brain Metastatic Colonization in a Model of Triple-Negative Breast Cancer and Induces DNA Double-Strand Breaks
Clin. Cancer Res., October 1, 2009; 15(19): 6148 - 6157.
[Abstract] [Full Text] [PDF]

Home page
 Mol Cancer ResHome page
D. Palmieri, D. Fitzgerald, S. M. Shreeve, E. Hua, J. L. Bronder, R. J. Weil, S. Davis, A. M. Stark, M. J. Merino, R. Kurek, et al.
Analyses of Resected Human Brain Metastases of Breast Cancer Reveal the Association between Up-Regulation of Hexokinase 2 and Poor Prognosis
Mol. Cancer Res., September 1, 2009; 7(9): 1438 - 1445.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
E. A. Maher, J. Mietz, C. L. Arteaga, R. A. DePinho, and S. Mohla
Brain Metastasis: Opportunities in Basic and Translational Research
Cancer Res., August 1, 2009; 69(15): 6015 - 6020.
[Full Text] [PDF]

Home page
 Ann OncolHome page
N. Cabioglu, A. A. Sahin, P. Morandi, F. Meric-Bernstam, R. Islam, H. Y. Lin, C. D. Bucana, A. M. Gonzalez-Angulo, G. N. Hortobagyi, and M. Cristofanilli
Chemokine receptors in advanced breast cancer: differential expression in metastatic disease sites with diagnostic and therapeutic implications
Ann. Onc., June 1, 2009; 20(6): 1013 - 1019.
[Abstract] [Full Text] [PDF]

Home page
 Molecular Cancer TherapeuticsHome page
A. Baschnagel, A. Russo, W. E. Burgan, D. Carter, K. Beam, D. Palmieri, P. S. Steeg, P. Tofilon, and K. Camphausen
Vorinostat enhances the radiosensitivity of a breast cancer brain metastatic cell line grown in vitro and as intracranial xenografts
Mol. Cancer Ther., June 1, 2009; 8(6): 1589 - 1595.
[Abstract] [Full Text] [PDF]

Home page
 Ann OncolHome page
B. C. Pestalozzi
Brain metastases and subtypes of breast cancer
Ann. Onc., May 1, 2009; 20(5): 803 - 805.
[Full Text] [PDF]

Home page
 JNCI J Natl Cancer InstHome page
B. Gril, D. Palmieri, J. L. Bronder, J. M. Herring, E. Vega-Valle, L. Feigenbaum, D. J. Liewehr, S. M. Steinberg, M. J. Merino, S. D. Rubin, et al.
Effect of Lapatinib on the Outgrowth of Metastatic Breast Cancer Cells to the Brain
J Natl Cancer Inst, August 6, 2008; 100(15): 1092 - 1103.
[Abstract] [Full Text] [PDF]

Home page
 Clin. Cancer Res.Home page
W. J. Kil, D. Cerna, W. E. Burgan, K. Beam, D. Carter, P. S. Steeg, P. J. Tofilon, and K. Camphausen
In vitro and In vivo Radiosensitization Induced by the DNA Methylating Agent Temozolomide
Clin. Cancer Res., February 1, 2008; 14(3): 931 - 938.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplementary Data
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Palmieri, D.
Right arrow Articles by Steeg, P. S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Palmieri, D.
Right arrow Articles by Steeg, P. S.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online