[Frontiers in Bioscience E2, 627-640, January 1, 2010]
Characterization of a novel, trastuzumab resistant human breast cancer cell line
Mark Barok1, Margit Balazs2,3,, Viktoria Lazar2, Zsuzsa Rakosy2,3, Eniko Toth1, Andrea Treszl2, Gyorgy Vereb1, Gail T. Colbern4, John W. Park4, Janos Szollosi1,5
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TABLE OF CONTENTS
1. ABSTRACT
HER2-positive breast cancers represent a distinct phenotype and are intrinsically more aggressive than HER2-negative tumors. Although HER2-targeted therapies have been rationally developed, resistance to these treatments represents a process understood poorly. There are few experimental models that allow studying the molecular mechanism of resistance. Our aim was to characterize a trastuzumab resistant breast cancer cell line (B585) that was established from an invasive ductal carcinoma. B585 grows only in immunodeficient mice as a xenograft. CGH and FISH were used to define cytogenetic alterations, gene-expression analysis and immunohistochemistry were applied to detect RNA and protein expression. By array-CGH focused amplifications were identified for C-MYC, EGFR, ErbB2, CCND1 and TOP2-A oncogenes. ErbB2 was co-amplified with TOP2-A. mRNA overexpression was detected for the amplified genes. ErbB2 protein was overexpressed and showed heterogeneous distribution. In summary, molecular cytogenetic analysis and expression profiling of B585 revealed several new alterations. Based on the experiments performed in SCID mice and the genotypic/phenotypic characteristics, this new in vivo breast cancer xenograft is a valuable model to investigate molecular mechanism of trastuzumab resistance.
2. INTRODUCTION
Deregulation of type I receptor tyrosine kinases (RTKs) plays a crucial role in the development of many cancers (1, 2). In humans, the widely studied type I RTKs, known also as the EGF receptor family, includes four members: ErbB-1, ErbB-2, ErbB-3 and ErbB-4. All members of the EGF family share significant homology and consist of an extracellular binding domain, a transmembrane lipophilic segment and an intracellular tyrosine kinase domain. Binding of receptor-specific ligands to ErbB-1, ErbB-3 and ErbB-4 leads to the formation of homo- and heterodimers, which result in transmission of signals controlling normal cell growth and differentiation. No soluble ligand for ErbB-2 has been identified, however, it has been shown that ErbB-2 is the preferred heterodimerization partner for ErbB-1, ErbB-3 and ErbB-4 (3-6).
Point mutation and overexpression of ErbB-2 can lead to cell transformation (7) and overexpression of ErbB-2 in breast and ovarian carcinomas correlates with poor patient prognosis (8). In vitro and transgenic models clearly support the hypothesis that ErbB-2 is involved in oncogenesis (9, 10). Furthermore, withdrawal of ErbB-2 in transgenic models leads to complete resolution of the breast tumors (11). This suggests that ErbB-2 overexpressing breast cancers are driven by ErbB-2 and has lead to numerous efforts to target ErbB-2.
Trastuzumab, also known as Herceptin and rhuMAb HER2, is a humanized antibody derived from 4D5, a murine monoclonal Ab, which recognizes an epitope on the extracellular domain of ErbB2. Its addition to standard chemotherapeutic regimens is associated with high response rates and increased survival (12-15). Only ErbB2 overexpressing tumors are responsive to trastuzumab-based treatment and patients are selected using assays of protein overexpression or gene amplification. Trastuzumab is effective alone, as well as in combination with other drugs, such as taxane and vinorelbine, in many, although not all, ErBB2 positive tumors (16). For the rest of the patients the trastuzumab treatment is ineffective, although the ErbB-2 gene is amplified and the protein is overexpressed (17). This indicates that other factors must play a role in the development of primary resistance to trastuzumab and need to be identified prior to administration of therapy.
Although the mechanisms underlying the action of trastuzumab are still not fully understood, several molecular effects have been observed: ErbB2 receptor downregulation and subsequent inhibition of its downstream PI3K-Akt signaling pathway (18, 19), induction of cell cycle arrest in G1 phase (20), inactivation of the mitogen-activated protein kinase and phosphoinositide-3-kinase pathways (21, 22) induction of apoptosis (23), PTEN activation (24), increase in HLA-I-restricted antigen presentation of ErbB2 (25). In addition in vivo models, trastuzumab decreased the microvessel density of breast cancer xenografts (26), and efficiently mediated antibody-dependent cellular cytotoxicitiy (ADCC) (27, 28).
Potential mechanisms of resistance to trastuzumab are even less understood, and thus identification of biomarkers associated with resistance would be extremely useful in optimizing treatment outcomes for HER2 positive patients. The possible mechanisms of primary or acquired trastuzumab resistance include: autocrine production of EGF related ligands (29), activation of insulin-like growth factor-I (IGF-I) receptor pathway (30), masking of the trastuzumab epitope by MUC4, a cell surface sialomucin (31, 32) or by hyaluronan (33), loss of PTEN function (24), impaired ADCC reaction (27, 34, 35).
Identification of molecular markers of potential resistance could be useful in determining non-responding patients even before start of trastuzumab treatment. While a lot of mechanisms are suspected of causing trastuzumab resistance, it is extremely important to have several models to investigate trastuzumab resistance. Furthermore, new model systems are needed, which will help to broaden our knowledge about the mechanisms of resistance, enabling us to develop drugs circumventing the trastuzumab resistance. Although trastuzumab resistant cell lines were developed from the originally trastuzumab-sensitive BT-474 cell line (36, 37), it seems important to develop cell lines which were established from the tumor of trastuzumab resistant patients. To the best of our knowledge to this time there is only one breast cancer cell line, which is a cell line of this kind (38, 39). In this study we characterize an ErbB2 positive breast cancer cell line (entitled B585) derived from a cancer patient with a trastuzumab resistant tumor. The B585 cells are able to grow only in immunodefficient SCID mice and the B585 xenografts are resistant to trastuzumab (40). The characterization was performed by applying immunohistochemistry, fluorescence in situ hybridization (FISH), chromosomal and array comparative genomic hybridization (cCGH and aCGH) and gene expression analysis using Affymetrix microarray platform. In addition to the overexpression of the ErbB2 protein, both integrin beta 1 and CD44 were overexpressed on B585 cells similarly to that observed on JIMT-1 trastuzumab resistant cell line. The B585 model may provide a suitable system to investigate the role of integrin beta 1 and CD44 in the trastuzumab resistance, especially in the resistance against trastuzumab-mediated ADCC.
3. MATERIALS AND METHODS
3.1. Patient clinical history
The primary breast cancer tissue used to develop the B585 xenograft was originated from a 63 year-old patient, who later developed metastasis and the patient metastatic disease was clinically resistant to trastuzumab treatment and chemotherapy. The tumor was estrogen receptor negative and ErbB2-positive high grade invasive ductal breast cancer. The patient underwent surgery with radical mastectomy and axillary lymph node evacuation (40). Approval to use the tumor cells for further investigation was obtained from the patient and local Ethical Committee prior to the initiation of cell culture.
3.2. Maintaining the B585 primary breast cancer xenografts in SCID mice
The B585 cells can be kept alive only in vivo as breast cancer xenograft in immunodeficient mice, but cannot be serially passaged in vitro as we described earlier (40). Tumor xenografts were harvested from donor mice, finely minced under sterile conditions, washed twice in cold Hanks' buffer, and approximately 200 micrograms of tumor tissue in 200 microliters Hanks' buffer mixed with equal volume of Matrigel (BD Matrigel, BD Biosciences, USA) was injected subcutaneously into the recipient, seven week-old female SCID C.B-17 scid/scid (originated from the laboratory of Fox Chase Cancer Center, Philadelphia, PA, USA) mice using a 18-gauge needle. The mouse population was housed in a pathogen-free environment. Only nonleaky SCID mice with murine IgG levels below 100 nanograms/mililiter were used in this study (41). Mice were monitored daily for general health. Tumor volumes were derived as the product of the length, width, and height of the tumor measured once a week with a caliper. Fifty days after inoculation the tumors started to grow and by day 90 the animals had to be euthanized because of the large tumor size. Tumor samples were collected for further analysis and the remaining tumor was transplanted into new animals.
Animals were euthanized by CO2 inhalation. The animal experiments were done with the approval of the Ethical Committee of the University of Debrecen.
3.3. Antibodies
The anti ErbB1 monoclonal antibody (mAb) 528 (IgG2a) was produced by the hybridoma cell line 528 (HB-8509, ATCC, Manassas, VA), the anti heavy chain of human class-I HLA A, B, and C mAb (W6/32) by hibridoma cell line W6/32 (kind gift of Francis Brodsky, University of California San Francisco, USA), the anti-integrin beta 1 mAb TS2 (IgG1) by the hybridoma cell line TS2/16.2.1 (HB-243, ATCC, Manassas, VA), the anti CD44 mAb Hermes-3 by the hybridoma cell line HB-9480 (ATCC, Manassas, VA). 528, TS2 and Hermes-3 antibodies were purified using protein A affinity chromatograpy. The anti ErbB2 mAb trastuzumab (Herceptin®) (IgG1) was purchased from Roche Ltd. (Budapest, Hungary). The mouse anti human epithelial specific antigen (ESA) (also known as EpCAM (epithelial cell adhesion molecule)) mAb (mouse IgG1) was obtained from Serotec (MCA1870; Serotec, UK). Alexa647-goat-anti-mouse IgG (GAMIG, H+L) was obtained from Molecular Probes (Eugene, Oregon, USA).
3.4 Conjugation of antibodies with fluorescent dyes
Covalent binding of AlexaFluor 488, AlexaFluor 546 and AlexaFluor 647 succinimidyl ester (Molecular Probes, Eugene, Oregon, USA) to the primary antibodies was carried out according to the manufacturer's instructions. The dye/protein labeling ratio was approximately 3:1.
3.5. Immunohistochemistry
Subcutan tumors were removed from anesthetized mice, covered with Shandon Cryomatrix (Thermo Electron Corporation, USA), and stored in liquid nitrogen. 20 micrometers thick cryosections were cut using a SHANDON AS-620E Cryotome (Thermo Electron Corporation, USA) on silanized slides. Samples were fixed in 4% formaldehyde for 30 minutes and were washed twice in PBS with 1% bovine serum albumin (BSA; pH, 7.4) for 20 minutes at room temperature. Slides were labeled with a saturating concentration (10-20 micrograms/mililiter) of dye-conjugated antibodies in 100 microliters PBS containing 1% BSA overnight on ice, washed twice with PBS. When indirect labeling scheme was used, cells were first incubated with the unlabeled primary antibody, followed by the dye-conjugated secondary antibody. Unbound antibodies were removed by washing twice with PBS. After labeling, the samples were covered with 15 microliters Mowiol (Merck Kft, Budapest, Hungary) to avoid bleaching of the fluorescent dyes.
3.6. Confocal microscopy
A Zeiss LSM 510 confocal laser-scanning microscope (Carl Zeiss, Göttingen, Germany) was used to image samples. AlexaFluor 488 was excited with the 488 nanometers line of an argon ion laser, and its emission was detected through a 505-530 nanometers bandpass filter. AlexaFluor 546 and AlexaFluor 647 were excited with the 543 and 633 nanometers lines of a green and a red He-Ne laser, respectively, and their emissions were measured between 560-615 nanometers and over 650 nanometers, respectively. Fluorescence images were taken as 1 micrometer optical sections using a 63x (NA=1.4) oil immersion objective.
3.7. Chromosomal and array comparative genomic hybridization (CGH)
Chromosomal CGH (cCGH) experiments were performed as described earlier (42). The results of hybridizations were evaluated using a digital image analysis system (ISIS, Metasystems GmbH, Altlussheim, Germany). Array CGH experiments and analysis were performed as described before (43) using Human array 3.1 array containing 2.464 overlapping bacterial artificial chromosome (BAC) (13) clones (44). Array-based CGH was performed as described by Pinkel et al. (45). The imaging setup and costume software were described by Pinkel et al. (45). Image analysis was performed using two software packages (SPOT version 1.2 and SPROC version 1.1 (46). Array validation was performed using normal human female reference DNA. Array spots with more than 0.25 Log2 values were considered as gains and less than - 0.25 Log2 values as losses.
3.8. Gene expression analysis
Total RNA was isolated from freshly removed B585 tissue using RNeasy Mini kit according to the protocol of the supplier (Qiagen, GmbH, Germany). Normal breast tissues were used as control. RNA concentrations were measured using NanoDrop ND-1000 UV-Vis Spectrophotometer (NanoDrop Technologies, Wilmington, Delaware USA). The integrity of the RNA was determined on the Agilent 2100 Bioanalyser using RNA 6000 Nano Kit (Agilent Technologies, Palo Alto, CA). All samples used for gene expression analysis had a 28S/18S ribosomal RNA ratio of more than 1.5. The mRNA levels were determined using Affymetrix GeneChip Human Genome U133 Plus 2.0 expression arrays (Heidelberg, GeneCore Genomics Core Facility, Germany). Gene expression analyses were carried out using the GeneSpring 7.3 software (Agilent Technologies). Raw intensity values from each chip were normalized to the 50 percentile of the measurements taken from that chip to reduce chip-wide variations in intensity. Each gene was normalized to the average measurement of that gene to enable comparison of relative changes in gene expression levels between different conditions. Volcano plot with a p value cut off of 0.05 were applied for identification of differentially expressed genes with minimum 2-fold differences. For multiple testing corrections the Benjamini and Hochberg false discovery rate was used.
3.9. Correlation of aCGH and expression data
The web-based MatchMiner program (47) was applied to match the amplified FISH clone IDs of aCGH to the appropriate Affymetrix microarray probe sets. For genes that were represented by more than one probe sets on the Affymetrix array, we used the sequence that gave the highest correlation between oligonucleotide array expression data.
3.10. Fluorescence in situ hybridization
For the characterization of breast cancer specific gene alterations at a single cell level double and triple labeled DNA probe sets were applied. The ProVysion multi-color probe kit consisted the centromere specific chromosome 8 (SpectrumAqua labeled), lipoprotein lipase region (LPL) (8q21.23) (SpectrumOrange labeled) and C-MYC (8q24) (SpectrumGreen labeled) (Vysis Inc. Des Plains, IL, USA). The chromosome 17 specific centromeric probe (SpectrumAqua) was cohybridized with Topoisomerase II-alpha gene (TOP2A, SpectrumOrange) and ErbB2 (SpectrumGreen) gene specific probes (Vysis Inc.). The N-MYC (2p24.1; SpectrumOrange) and the centromeric probe for chromosome 2 (Spectrum Green) as well as the EGFR (7p13; SpectrumOrange) and the centromeric 7 probe (SpectrumGreen) were hybridized simultaneously (Vysis Inc.). The 8p22 region specific probe was isolated from bacteria clones generously supplied by Molecular Genetic Resources (University of Bari, Bari, Italy). Centromere-specific probes for chromosomes 1, 2, 6-9, 11, 12, 17, 18, 20 and X were isolated from bacteria clones (University of Bari, Italy). DNA probes were labeled with biotin-dUTP by nick translation using BioNick kit, according to the protocol of the supplier (Gibco BRL, Germany). All FISH experiments were carried out as described earlier or according to the manufacturer instructions (48-50). Samples were scored for the number of fluorescent signals in each nucleus by using a fluorescence microscope (OPTON, Oberkochen, Germany) equipped with selective filters for the detection of FITC, SpectrumGreen, SpectrumOrange and DAPI. Approximately 200-500 nuclei and/or 10 metaphases were scored for each hybridization. Three-color images were captured using a digital imaging analysis system (ISIS, Metasystems GmbH, Althussheim, Germany).
4. RESULTS
4.1. Immunohistochemical characterization of the B585 xenograft
Similarly as previously reported (40) our attempts to develop in vitro cell culture from the B585 xenografts using different cell culture conditions were unsuccessful. The B585 cells can recover from cryopreservation, and can grow only in immunodeficient mice as xenograft and they can be bred as transplantation into new animals. Injection of B585 cells subcutaneously into SCID mice lead to formation of xenograft tumors in more than 90% of the animals tested. From the seventh week onwards, the tumors started to grow exponentially in these mice and by day 90 the animals had to be euthanized because of the large tumor size. Fast frozen tissue sections were labeled with fluorescent antibodies. Figure 1. A, D, G show the positive immunohistochemical staining of the B585 tumor cells for the HLA-I antigen. Similarly to the original primary tumor the B585 cells maintained in xenograft were intensely ErbB2-positive (Figure 1 B, E, H, J). The established xenograft cell line was also positive for CD44 (Figure 1C), ErbB1 (Figure 1F), integrin beta 1 (Figure 1I) and ESA (Figure 1K).
4.2. Genetic alterations in the original primary breast tumor and the B585 xenograft
To search for copy number alterations in the B585 genome, we performed chromosomal CGH analysis using DNA from both samples (patient's primary tumor and xenograft). Copy number alterations of the original tumor and the B585 cell line matched well, 24 aberrations were seen by cCGH in the original lesion and B585 xenograft as well. Common alterations involved 11 chromosomal region gains and 9 regional deletions. Alterations present in both samples included gains at 1q, 2p11.2-p15, 6p11.2-p21, 6p22-p23, 7, 10p, 11q12-q14, 12p, 16p, 18q11.2-q21, 19 and losses at 2p16-pter, 3p14-q13.3, 4p, 4q12-q21, 4q22-qter, 8p21-8pter, 13q, 14q, 15q11.2-q22.2. The cCGH data are summarized in Table 1.
In order to increase the resolution of copy number gains and losses array CGH analysis was also performed using the 1.4 MB BAC array (HumArray3.2). The array CGH results were highly concordant for regions identified by cCGH, however numerous single copy amplicons and small deletions, that were not seen by cCGH were also detected. The quality of the array CGH experiments were tested using normal versus normal hybridizations, which were performed simultaneously with the xenograft sample hybridization. On the basis of the control experiments, we used log2ratio threshold of more than 0.25 for DNA copy number gains and log2ratio less than - 0.25 for DNA copy number losses. High level copy number alterations (defined as log2ratio more than 1) were seen on 34 (Table 2), including the LETM2, FGFR1 (8p12-8p11.23), C-MYC (8q24.21), CCND1 (11q13.2-11q13.3), ErbB2 (17q12) oncogenes, whereas homozygous deletions (defined as log2ratio less than or equal to 1) were observed on 52 clones Table 3. Most of these deletions were seen on 8p21-8p23 (26 clones), on 14q31.11-14q32.14 (11 clones) and on 15q21.1-15q21.3 (6 clones). Figure 2 shows the whole genome aCGH profile of the B585 genome. High level amplification exceeding log2 mean raw ratio more than 2 was present only in a few clones. In order to validate the copy number alterations observed by array CGH interphase FISH was applied using different combination of centromeric and locus specific probes.
4.3. Fluorescence in situ hybridization using centromere and gene specific probes
The ploidy pattern of the B585 cells were determined using 13 different centromere specific probes (chromosomes 1, 2, 5- 9, 11, 12, 17, 18, 20 and X). Chromosome index (CI: total number of centromeric signals/number of nuclei counted), which is closely related to the DNA content of the tumor, was calculated as 3.35 (Table 4). High level amplification was detected for the C-MYC (8q24.21), CCND1 (11q13.2-11q13.3), ErbB2, TOP2A (17q12) and intermediate amplification for the EGFR (7p12) and EGR1 (5q34) oncogenes. Interestingly only one or two copies (in relation to the chromosome 2 centromere) were deleted of the N-MYC (2p23-24) oncogene in the majority of cells. The deletion was also seen by aCGH. Summary of the FISH and aCGH data are presented for the studied regions in Table 5.
Chromosome 8 copy number alterations were greatly complex. High level amplification was seen on the 8p11.2 (clones: RP11-48D21, RP11-282J24, RP11-73M19) covering the FGFR-1 gene (Fibroblast growth factor receptor-1 gene) and on 8q24.1, 8q24.12-q13 and 8q24.2 which harbor the C-MYC oncogene. Deletion of the proximal part of the short arm (8p12-pter) was observed (Figure 3A). To validate the deletions at the 8p12 region and the amplification at the 8q24 loci we performed FISH analysis at a single cell level using tricolor FISH, containing the CEP® 8 probe labeled with SpectrumAqua, lipoprotein lipase (LPL) labeled with SpectrumOrange and the C-MYC gene labeled with SpectrumGreen (Figure 3C). A separate experiment was performed using the 8p22 probe simultaneously with chromosome 8 centromere. The interphase FISH results are summarized on Figure 3B. Chromosome 8 centromere were present mainly in three copies (66%), whereas the 8p22 locus was deleted in more than 75% of cells, the C-MYC oncogene was amplified in 55% of cells (amplification level is approximately 4 fold relative to the centromeric 8 signals) and the 8p22 locus was deleted in more 65% of cells.
Out of the 192 BAC clones covering chromosome 11 nineteen were amplified at the 11q13-q14 region. By using FISH we could detect high copy number amplification of the CCND1 gene. The amplification pattern of CCDN1 was heterogeneous; there was a significant fraction of cells having more than 20 signals.
Figure 4A shows the aCGH profile of chromosome 17 of the B585 xenograft. Two regions were amplified on chromosome 17, one is the ErbB2 (17q12) and the second is 17q24-25 loci containing the MAP2K6 (mitogen-activated protein kinase kinase 6: 17q24.3) and the GRB2 (growth factor receptor-bound protein 2: 17q24-25) genes. Using chromosomal CGH we could not detect gain on 17q12 (Table 1.), however array CGH showed clear amplification of this loci. The amplification of the 17q12 was validated by FISH with three color probe set (LSI® TOP2A SpectrumOrangeTM /HER-2 SpectrumGreenTM CEP® 17 SpectrumAquaTM), Figure 4B illustrates the distribution of these genes and the Figure 4C demonstrates a representative FISH image clearly showing the coamplification of ErbB2 and TOP2A on the magnified image part: green fluorescent spots (ERbB2 signals) are frequently very close to the red spots (TOP2A).
4.4. Gene expression signature of the B585 xenograft
Gene expression signature of the B585 cells were defined on Affymetrix GeneChip Human Genome U133 Plus 2.0 expression arrays. After the normalization and quality control steps Volcano plot p-value cutoff 0.05 with Benjamini and Hochberg False Discovery Rate was applied to identify genes differentially expressed (more than 2 - fold) between the B585 cells and control sample. Affymetrix gene expression analysis resulted in 564 differentially expressed genes, raging from 145-fold increased in odontogenic ameloblast-associated protein to an 82.8-fold decrease in SPARC-like 1. Out of these genes 416 were found to be upregulated and 148 downregulated in B585 cells versus normal breast sample. Overexpression of different genes was detected on all chromosomes. Correlation of gene expression level and gene amplification was also performed. Out of the 44 amplified genes we could identify 37 different transcripts on the Affymetrix Human 2 array (Table 6). From these thirty-seven genes we found only three (clone ID CTC-298G6 on 12p12.1, RP11-53C3 on 12p13.32 and RP11-122P17 on 16q24.1 regions) that were down regulated when compared to normal breast tissue expression level. The larger fraction (65%) of the amplified genes was overexpressed more than 2 - fold. The highest fold change was detected for the GINS2 (16q24.1) and ErbB2 genes. Relatively high fold change was noticed on genes that were amplified around the CCDN1 gene on chromosome 11q13.2-q13.3. Near this region, the expression level of the amplified PAK1 serine/threonine kinase was also increased. The mRNA level of the C-MYC genes was less prominent when compared to the normal sample, the fold change for this gene was less than 2. When we compared the expression level of genes that were selected based on their association with trastuzumab treatment response in human breast tumors, we found that the CCNE1, CDK1 and ErbB2 genes have the highest expression level (exceeding more than 8 fold over mean gene expression of the controls), other genes including the SKP2, FLNA, ITGB4, PRKDC, ERbB3, COP1, RELA, GRB7, RARA and FADD genes were also highly upregulated. Tumorsuppressor genes including PTEN, JAK1, MAPK1, TP53 ESR1, LYN, JUN and CAV2 genes were found to be downregulated in the B585 xenograft.
5. DISCUSSION
We report here the cytogenetic and immunohistochemical characterization of a human breast cancer cell line (B585) that was established from an invasive ductal breast carcinoma of a 63 year-old patient who was resistant to trastuzumab treatment (40). The B585 cells can grow only in immunodeficient mice, and the B585 xenografts are resistant to trastuzumab treatment similarly to the primary tumor (40).
Using chromosomal CGH we could compare the copy number alterations of the original tumor and the xenograft and found that most of the alterations were similar, which is a sign for the common clonal origin of lesions. This observation indicates that the cell line retained most of the genetic characteristics of the parental tumor. The divergence between the two samples can be explained in different ways: i.) the genetic make up of tumor cells in the xenograft are heterogeneous, but the ratio of cells exhibiting the different alterations is higher in the B585 xenograft than it was in the primary lesion, ii.) the normal cell contamination of the primary breast cancer tissue could mask some alterations present in a small fraction of cells, thus it may cover less dominant changes in a genetically heterogeneous tumor, in addition those cells can have higher proliferative rate in the xenograft, iii.) it can not be excluded either that alterations seen only in the cell line are the results of in vivo cell culturing.
Because the resolution of cCGH is limited, to more precisely describe and map the chromosome copy number alterations we also performed array CGH analysis on B585 cells. The results of cCGH and aCGH analyses were highly concordant for the main regions identified by cCGH, however numerous single copy amplicons and small deletions, that were not seen by cCGH were also discovered by aCGH. High level of copy number alterations that are predictive for poor prognosis of breast cancer patients were all present in the B585 xenograft, those included amplifications on chromosome 8, 17 and 20 (51). The cytogenetic alterations detected by array CGH in the B585 genome are similar to the JIMT1 cell line, a recently described trastuzumab resistant cell line (51). Complex pattern of genomic alterations were detected in B585 on chromosomes 2, 4, 8 as well as chromosome 11q13, 16q and 17q. The highest level of amplification was seen on the 8p12-8p11.23 region which covers many genes including the FGFR1 (fibroblast growth factor receptor-1 (fms-related tyrosine kinase-2)) putative oncogene. It was found by others that chromosome 8p12-8p11.23 locus is amplified in 10-15% of human breast cancers (52). Amplification of this region has been associated with estrogen receptor-positive tumors and lobular histology (53). The FGFR1 is excluded from the amplicon in some tumors (54). There is less information available about the role of highly expressed genes on this locus, including LETM2, DDHD2, WHSC1L1 and PPAPDC1B which were overexpressed in B585 between 4 to 9 fold when compared to the expression values to normal breast tissue. Relatively high level amplification of the C-MYC was detected by aCGH which was also confirmed by FISH, the C-MYC and chromosome 8 ratio was 4 in more than 50% of cells and was above 6 in 12% of B585 cells. It has been shown that the C-MYC amplification was significantly associated with high histological grade, negative progesterone receptor status DNA aneuploidy, high S-phase fraction and with poor distant metastasis-free survival (55). However, we have to note that the amplification of the C-MYC oncogene in the B585 cell line was not associated with high mRNA level.
Chromosome locus 11q13 is frequently amplified in a number of human cancers including carcinoma of the breast (56, 57). Originally 11q13 amplification was thought to involve only a single amplicon spanning many megabases, but more recent data have identified four core regions within 11q13 that can be amplified independently or together in different combinations. The present CGH array data support this observation. The clustered amplifications on chromosome 11q13-14 region pin point that numerous genes are amplified around the CCND1 gene. This region harbors several genes with known or suspected oncogenic potential, the complex structure of the amplicons and the fact that 11q13 is gene-rich locus have contributed to the identification of specific genes that contribute breast cancer tumorigenesis and make it a difficult and continuing process. To date CCND1 (encoding the cell cycle regulatory cyclin D1) and EMS1 (encoding the filamentous actin binding protein and c-Src substrate cortactin) are the favored candidates (58).
Several reports have documented that close to the nearby TOP2A gene is often coamplified or deleted in breast carcinomas with ErbB2 amplification, and recent finding suggests that coamplification plays an important role in facilitating a favorable response to antracyclines, which target topoisomerase IIa (59). Another report has shown that several genes between ErbB2 and TOP2-A are often coexpressed in breast carcinoma cell lines with amplification of the ErbB2 gene. This opens the possibility that these other genes may play an important role in determining malignant potential and response to therapy, including trastuzumab.
Affymetrix gene expression analysis showed differentially upregulated and downregulatred genes. The set of highly expressed genes includes many genes with functional roles in cell cycle regulation and proliferation such as CDKN2B, CDC23, MCM8 and RFC2 as well as genes that are involved in cytokine-cytokine receptor interactions such as the CCL19, VEGFA genes. Increased expression levels of TOP2A and ErbB2 genes were detected. While the expression levels of ErbB3 protein (the preferred and most potent dimerization partner of ErbB2 (60) and ErbB4 are low on B585 cells (our unpublished observations), the ligand-less ErbB2 may recruit ErbB1, (which is overexpressed in B585 cells, Figure 1F), to boost its signaling potential through ErbB1. Recently, the involvement of ErbB2 in non-ErbB protein-mediated signaling has been brought into the focus of cancer research. Among these, beta integrins and CD44 are probably the best known candidates whose roles in cancer progression are well documented (61-64). In addition, CD44 is also involved in the direct regulation of both ErbB2 and ErbB1 (63). Furthermore, while ADCC had a key role in the tumor inhibitory effect of trastuzumab in vivo (27, 65), and CD44 was also suggested being able to prevent the recruitment of Fc receptor bearing immune effector cells, the B585 model (we showed that both integrin beta 1 and CD44 are overexpressed on B585 cells) may provide a suitable system to investigate the role of integrin beta 1 and CD44 in the trastuzumab resistance, especially in the resistance against trastuzumab-mediated ADCC. Furthermore, we found that epithelial specific antigen (ESA), a cell surface molecule that is known to be highly expressed in primary and metastatic breast cancer (66, 67) and together with CD44 have also been identified as prospective marker for breast cancer stem cells (68, 69) are also overexpressed on B585 cells.
In summary we have described a detailed characterization of a recently established trastuzumab-resistant breast cancer xenograft, B585. The B585 cells exhibit chromosomal alterations that are characteristic for breast tumors with a highly aggressive phenotype. The copy number alterations of the original primary tumor and the xenograft are similar. Beside known alterations numerous new copy number changes were identified. The B585 xenografts are resistant to trastuzumab administration, in spite of that the B585 cells are heterogeneously amplified for the ErbB2 gene and carry a high level mRNA and an intermediate protein expression for ErbB2, those histological features are representative for highly invasive trastuzumab-resistant ductal breast carcinoma that makes the B585 cells suitable model to study trastuzumab-resistance in model experiments and to use to assess potential therapies for this type of breast tumors.
6. Aknowledgement
Mark Barok and Margit Balazs contributed equally to this work. The study was supported by the following grants Hungarian Academy of Sciences (2006TKI247), Hungarian Scientific Research Fund, OTKA T 048750, T 75191, T 68763, K62648, 75752, University of Debrecen, Hungary Mecenatura 1620 29/0017/1/29, NIH P50 CA58207, FIRCA R03 TW 00871-01A2, EU FP6 LSHB-CT-2004-503467, LSHC-CT-2005-018914, MCRTN-CT-035946-2, and MRTN-CT-2005-019481.
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Abbreviations: aCGH: array comparative genomic hybridization; cCGH: chromosomal comparative genomic hybridization; FISH: fluorescence in situ hybridization; SCID: severe combined immunodeficiency disease; BAC: bacterial artificial chromosome; IgG: immunoglobulin G; GAMIG: goat-anti-mouse immunoglobulin G; ESA: epithelial specific antigen; RTKs: receptor tyrosine kinases; mAb: monoclonal antibody; EGF: Epidermal Growth Factor; ADCC: antibody-dependent cellular cytotoxicitiy;
Key Words: Drug Resistance, Trastuzumab, Breast Tumor Xenograft, ErbB2/HER-2, array-CGH, Gene And Protein Expression
Send correspondence to: Margit Balazs, Division of Biomarker Analysis, Department of Preventive Medicine, Faculty of Public Health , Medical and Health Science Center, University of Debrecen, 4028 Debrecen Kassai str. 26/b, Hungary, Tel: 36-52-460-190/77151, Fax: 36-52-417-267, E-mail:margo@med.unideb.hu