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Approximately 25% of breast cancers overexpress the ErbB2 (HER2) receptor, and this receptor is targeted for treatment, e.g. by Trastuzumab, an anti-HER2 monoclonal antibody and/or by Lapatinib a tyrosine kinase inhibitor of EGFR (HER1) and HER2. Over time some tumours that initially respond to these treatments develop resistance; this may be a manifestation of robustness, i.e. the cells adapting to perturbations in their environment. Three HER2-positive cancer cell lines, HCC1954, SKBR3 and BT474 and their respective Lapatinib resistant variants (developed in the School of Pharmacy and Pharmaceutical Sciences, Trinity College Dublin) as HCC1954 RL (resistant to Lapatinib), SKBR3 RL (resistant to Lapatinib) and BT474 RL (resistant to Lapatinib) were studied for this project.
Characterisation of three cancer cell lines pairs, Control and RL was performed. Invasion and migration assays were performed on HCC1954 and SKBR3 sensitive and resistant pairs. Increases of 25.7% in invasion and 84.4% in migration of SKBR3 RL cells compared to SKBR3 parent cells were observed; whereas the HCC1954 cell line pairs were seeded too densely to note any differences.
RNA was isolated from all three cell lines pairs and subsequently quantified using NanoDrop. The levels of expression of a number of genes were measured in both Control and Lapatinib resistant cell lines. The gene transcripts selected for analysis were AXL, EGFR, ERBB3, FOXO3, IGF1R, PTEN, Sample A* and Sample B*, and the levels of expression for the Lapatinib-resistant compared to the control cells. (*Sample A and Sample B gene transcripts and their assays are in the process of being patented, so for IP protection their actual names cannot be listed in this report). This was to determine if these genes were up- or down- regulated in the Lapatinib resistant cell lines. Overall, EGFR and Sample B mRNAs were most substantially up-regulated and AXL most substantially down-regulated.
Abbreviations: PBS - phosphate-buffered saline, EGFR - epidermal growth factor receptor, EGFRl - epidermal growth factor receptor 1, HER - human epidermal growth factor receptor, HER2 - human epidermal growth factor receptor 2, FBS - foetal bovine serum, qRT-PCR - quantitative real time polymerase chain reaction, DNA - Deoxyribonucleic acid, cDNA - complementary DNA, RNA - Ribonucleic acid, mRNA - messenger RNA, miRNA - micro RNA, DMSO - Dimethyl sulfoxide, EDTA - Ethylenediaminetetraacetic acid, RL - Resistant to Lapatinib, ECM - Extra-cellular matrix, PBS - Phosphate buffered solution, RPMI - Roswell Park Memorial Institute, erbB - erythroblastic leukemia viral oncogene homolog, TKI - Tyrosine Kinase Inhibitor, MMLV - Moloney Murine Leukaemia Virus
The Epidermal Growth Factor Receptor (EGFR) or ErbB family of receptors consists of four structurally-related receptor proteins, EGFR1 (HER1 or erbB1), HER2 (neu or erbB2), HER3 (erbB3) and HER-4 (erbB4), with each having an extracellular ligand binding domain; a single transmembrane domain; and a cytoplasmic tyrosine kinase domain (Browne et al 2009). These four receptors are involved in many cellular responses, HER2 can bind with itself (homodimerise) or the other three proteins within the family to form heterodimers, and has the strongest kinase activity. Thus aberrations in HER signalling, particularly HER-2, have been implicated in the progression of breast cancer. According to the American Cancer Society about 180,000 new cases of breast cancer are diagnosed each year. Approximately 8,000 to 10,000 women die from metastatic HER2 positive breast cancer each year.
When epidermal growth factor and its relatives bind the ErbB family of receptors, they trigger a rich network of signalling pathways, culminating in responses ranging from cell division and growth to apoptosis, motility or migration to adhesion (Yarden & Sliwkowski, 2001). Ligands bind to the extracellular domain promoting dimerisation, resulting in the intracellular tyrosine kinase domain becoming activated. Jones et al (2006) studied the four receptors, but had to exclude HER4 due to lack of data. They noted that at higher expression levels, EGFR and HER2 become much more promiscuous than when their expression levels are low; whereas HER3 does not. Furthermore, the recruitment sites on HER2 are much more promiscuous than those on the other receptors. Amplification of EGFR and HER2 is frequently observed in human cancers and they propose that the elevated oncogenic properties of EGFR and HER2 are due, in part, to their ability to activate different pathways when overexpressed, and not just by increasing their primary pathway signalling.
Brown et al (2009) noted that HER receptors are widely expressed and functionally important in multiple tissues, particularly in mammalian development, with a role identified for EGFR in regulating epithelial development. EGFR-deficient mice have shown impaired epithelial development in several organs including skin, lung, and gastrointestinal tract; with brain, kidney and liver abnormalities resulting in death within weeks of birth. Mutations in HER2, HER3 or neuregulin-1 proteins resulted in a lack of neural crest precursor cells and mouse embryos lacking HER2, HER3, HER4 or neuregulins died during embryogenesis, due to severe cardiac abnormalities and brain defects. HER2 is also expressed in adult cardiomyocytes, and loss of cardiac HER2 can cause cardiomyopathy in adult mice - which has important clinical implications for HER2 targeted therapies. Overexpression of HER2 and subsequent dimerisation and phosphorylation activates Akt, MAPK and PKC signalling pathways, which ultimately promote proliferation and cell survival (see Figure 1A).
Figure 1A - HER 2 signalling pathway (Brown, et al, 2009)
Figure 1B - Inhibition of HER-2 signalling:
Trastuzumab binds extracellular domain of HER-2 dimers reducing HER-2 signalling;
Lapatinib inhibits tyrosine kinase domain of HER-2 and EGFR
A universal trait of complex biological (and non-biological) systems is their capacity to maintain function despite external and internal perturbations, a characteristic referred to as robustness (Bublil and Yarden, 2007). In this review the authors mention that perturbation of the ErbB function is implicated in some human diseases, ranging from reduced expression and signalling in some neurodegenerative diseases to overexpression and increased signalling in many cancers. High levels of EGFR expression were found in the majority of carcinomas, and amplification of the HER 2 gene can be found in 20-30% of metastatic breast lesions. Therefore, the ErbB receptors are attractive candidates for targeted therapy and, to date, several anti-ErbB monoclonal antibodies (e.g. Trastuzumab or HerceptinÂ®) and small-molecule tyrosine kinase inhibitors (TKIs e.g. Lapatinib or TykerbÂ®) have been licensed. The exact mechanism(s)-of-action of these therapies is not fully understood, nor is the method by which the cancer cells develop resistance to them. Bublil and Yarden (2007), however, propose that resistance to ErbB-targeted therapeutics could be viewed as manifestations of robustness.
Trastuzumab is a humanised monoclonal antibody that targets HER2, and was approved by the FDA in 1998 for the treatment of patients with metastatic breast cancer whose tumours overexpress the HER2 protein and who had received one or more chemotherapy regimens for their metastatic disease. Trastuzumab (in combination with paclitaxel) was indicated for treatment of patients with metastatic breast cancer whose tumours overexpress HER2 protein and who have not received chemotherapy for their metastatic disease. Trastuzumab is a large protein molecule that targets the part of the HER2 protein on the outside of the cell (see Figure 1B). In 2007 the FDA approved Lapatinib a Tyrosine Kinase Inhibitor (TKI), to be used in combination with capectabine (XelodaÂ®), another cancer drug, for patients with advanced metastatic breast cancer that is HER2 positive. The combination treatment was indicated for women who had received prior therapy with other cancer drugs, including an anthracycline, a taxane, and Trastuzumab (HerceptinÂ®). Lapatinib is a small molecule that enters the cell and blocks the function of this and other proteins and works through multiple pathways to deprive tumour cells of signals needed to grow (see Figure 1B). An advantage of Lapatinib is that it is in tablet form that the patient can take in their home, while Trastuzumab must be administered by infusion in a clinical setting.
Materials & Methods
A class II down-flow re-circulating laminar flow cabinet was used for cell culture work and aseptic techniques were employed at all times. 70% ethanol was used to swab the laminar flow cabinet before and after use and any items to be used in it. Air was allowed to circulate in the cabinet for 15 minutes between cell lines, and only one cell line was worked on at a time.
Lapatinib Tosylate was obtained in powder form from Sequoia Research Products Ltd. (SRP01211l). The Lapatinib powder was made up as stock solution in DMSO at a concentration of 10mM. A working concentration of 1:1000 was made up fresh weekly and kept at 4°C. The working concentration was used for assays and treatment of cell lines.
Three breast cancer cell lines were used; HCC1954, SKBR3 and BT474. The isolation and culturing of human HCC1954 (Gazdar AF, et al, 1998), SKBR3 (Trempe, 1976) and BT474 (Lasfargues EY, et al., 1978) cells have been detailed in previous studies.
Each cell line was grown as a monolayer in 25cm3 culture flasks in an incubator at a temperature of 37°C and 5% CO2. Cells were cultured in RPMI medium containing 10% Foetal Bovine Serum (FBS) and 1% L-Glutamine. Cells were subcultured and their medium renewed every two to three days. The cells were monitored for contamination prior to cell culture. To trypsinise/passage, culture medium was removed from the flasks and discarded. 2ml of Trypsin/EDTA solution was added and the flask was then incubated at 37°C for approximately 2 minutes until all the cells were seen to have detached. 2ml of growth medium was then added to neutralise the trypsin. This solution was transferred to a 30ml sterile universal tube and centrifuged at 900rpm for 5 minutes. The cell pellet was re-suspended in 4ml fresh growth medium and cells were counted using a haemocytometer to re-seed at the correct density into a fresh flask for subsequent RNA isolation.
Lapatinib-resistant cell lines were established in the laboratory (the School of Pharmacy and Pharmaceutical Sciences) over a period of five months, by stepwise exposing to increased concentrations of Lapatinib, from 10nM to 250nM; which eventually became the maintenance dose. Three new Lapatinib -resistant cells lines were thus established; HCC1954 RL (Resistant to Lapatinib), SKBR3 RL (Resistant to Lapatinib) and BT474 RL (Resistant to Lapatinib). Parent cell lines were "aged" as controls for the respective RL cell variants, by culturing in parallel, but without exposing to Lapatinib.
In Vitro Invasion Assay
The invasive capacity of the HCC1954 Control, HCC1954 RL, SKBR3 Control and SKBR3 RL cell lines was investigated using a slightly modified invasion assay protocol (O'Driscoll et al, 2002). This assay was carried out once (with two samples), simultaneously for each of the two cell lines (control and RL). This was performed by coating the transwell inserts with an ECM (Sigma) 24 hours prior to use. The cell suspension (5x105 cells/insert) with 1% FBS medium was placed inside the insert and 10% FBS medium in the well. Cells were incubated at 37°C for 48 hours for HCC1954 and 72 hours for SKBR3 (due to HCC1954 having a higher proliferation rate).
After this time had elapsed, the inner side of the insert was wiped with a PBS-soaked swab in order to remove any cells which had not migrated through the insert. The inserts were gently rinsed with PBS three times and were stained, for ten minutes, using 25% crystal violet. This was then rinsed with PBS and allowed to air-dry.
Inserts were then viewed using light microscopy and cells, at four- and ten-magnification, were examined and photographed. In order to quantify the cells stained with crystal violet, 200Î¼l of 33% glacial acetic acid was added to solubilise the crystal violet. 50Î¼l of the eluted stain was transferred from each well into individual wells of a 96 well plate and the absorbance was read at 570nm using a plate reader (FLUOstar OPTIMA-Fluorescence Microplate Reader).
In Vitro Migration Assay
The migration ability of the HCC1954 Control, HCC1954 RL, SKBR3 Control and SKBR3 RL cell lines was investigated. The protocol for the motility assay was identical to that outlined above for the invasion assay, with the exception that the inserts were not coated with ECM. This assay was carried out once (5x105 cells/insert) simultaneously for the cell lines. Again HCC1954 cells were incubated at 37°C for 48 hours, and SKBR3 cells for 72 hours.
RNA Isolation from Cells
Isolation of RNA from cells was performed using an 'RNeasy Mini Kit (50)' (Qiagen Cat. No. 74104) using the manufacturer's protocol.
RNA Quantification using NanoDrop Spectrophotometer
The amount of RNA in each sample cell line was quantified using the NanoDrop Spectrophotometer. The ND-1000 software automatically calculated the quantity of RNA in the samples using the OD260 reading. RNA has its absorption maximum at 260nm, i.e. OD260 x 40 x Dilution Factor/1000 = RNA content (Âµg/Âµl).
The software simultaneously measured the OD280 of the samples, allowing the purity of the sample to be estimated: Purity = OD260/OD280, and a ratio of ~2.0 is generally accepted as "pure" for RNA. If the ratio is appreciably lower, it may indicate the presence of protein, phenol or other contaminants that absorb strongly at or near 280 nm.
The OD260/OD230 ratio was also assessed, giving an indication of the integrity of the RNA sample. The 260/230 values for "pure" nucleic acid are often higher than the respective 260/280 values. Expected 260/230 values are commonly in the range of 2.0-2.2. If the ratio is appreciably lower than expected, it may indicate the presence of contaminants which absorb at 230 nm.
Reverse-Transcription Polymerase Chain Reaction Analysis
Reverse Transcription of RNA from cells (cDNA Synthesis)
mRNA was transcribed to cDNA by a 2 step reverse transcription process. 800ng of RNA was used to prepare the cDNA, as summarised in Table 1.
Once the RNA mixture had cooled (~2 min.), 10Âµl of the master mix was added and mixed by flicking. The mixture was centrifuged to collect the material in the bottom of the tube and then incubated at 37Â°C for 1 hr. 1:10 dilutions in RNase-free water were prepared for the cell cDNA and these were stored at -20Â°C.
Quantitative Real Time Polymerase Chain Reaction (qRT-PCR)
The cDNA was analysed for the expression of genes of interest by qRT-PCR (as summarised in Table 3). The genes of interest were selected from a whole genome microarray study performed, in this laboratory, on SKBR3 Control and RL cells. TaqMan probes are oligonucleotides that are designed, based on nucleotide make-up, to be specific for the transcript of interest, have fluorescent reporter dyes attached to the 5' end and a quencher moiety coupled to the cDNA region to be amplified by these primers. These probes are designed to hybridize to an internal region of a PCR product. In the unhybridised state, the proximity of the fluor and the quench molecules prevents the detection of fluorescent signal from the probe. During PCR, when the polymerase replicates a template on which a TaqMan probe is bound, the 5'- nuclease activity of the polymerase cleaves the probe. This decouples the fluorescent dye, thus increasing the fluorescence in each cycle, proportional to the amount of probe cleavage. In order to exclude any amplification product derived from genomic DNA or any other contaminant that could contaminate the RNA preparation, total RNA without reverse transcription was used as a negative control. Water, on its own, replaces RNA as a negative control to rule out presence of any contaminating RNA or DNA in the other components of the reaction mix. The PCR cycle was performed, as summarised in Table 3.
RNA and RNase and DNase-free H20
The mix was incubated at 70Â°C for 10 mins.
Table 1: Components of the cDNA Reaction Mixture
RNase and DNase-free H20
10 Î¼l was added to each well and incubated for 37Â°C for 50 mins.
Table 2: Components of the qRT-PCR Reaction Mixture
CYCLE (40 cycles)
Table 3: Thermal cycling conditions used in this study
Statistical Analysis of Data
Comparative statistical analysis was not carried out on the quantitative data generated in this study as all assays were not carried out in triplicate (due to time limitations) and, therefore, statistical analysis would not be meaningful and may be misleading.
RNA Quantification using NanoDrop Spectrophotometer
RNA was quantified to determine the correct amount for cDNA transcription.
SKBR3 CON R-1
SKBR3 LAP 250 R-1
HCC1954 CON R-1
HCC1954 LAP 250 R-1
SKBR3 CON R-2
SKBR3 250 LAP R-2
BT474 CON R-1
BT474 LAP 250 R-1
HCC1954 CON R-2
HCC1954 250 LAP R-2
SKBR3 CON R-1
Table 4: RNA Concentrations measured by NanoDrop Spectrophotometer
Invasion and Migration Assays
As outlined above, the SKBR3 cell line grew for 72 hours before being examined and photographed and the cells quantified by plate reader. There was a noted difference in both the invasion and migration assays between the Control and the RL variants (see Figure 2).
SKBR3 Inv CTRL2-1.jpg
SKBR3 Inv LAP 2-2.jpg
SKBR3 Mig CTRL 1-1.jpg
SKBR3 Mig LAP 1-1.jpg
SKBR3 Control Invasion
SKBR3 Lapatinib Resistant Invasion
SKBR3 Control Migration
SKBR3 Lapatinib Resistant Migration
Figure 2. Control and RL cells Invasion and Migration as evident by microscopy (above) and following dye elution and quantification (bottom).
The HCC1954 cell line grows more rapidly so, although these were seeded for only 48 hours, the inserts were almost confluent with cells and there was little visible or measurable difference (see Figure 3).
HCC1954 CTRL 1-2.jpg
HCC1954 LAPr 2-2.jpg
HCC1954 Mig CTRL 1-2.jpg
HCC1954 Mig LAPr 1-2.jpg
HCC1954 Control Invasion
HCC1954 Lapatinib Resistant Invasion
HCC1954 Control Migration
HCC1954 Lapatinib Resistant Migration
Figure 3. Control and RL Invasion and Migration as evident by microscopy (above) and following dye elution and quantification (bottom).
Quantitative real time Polymerase Chain Reaction (qRT-PCR)
Results for the various gene transcripts are graphed below. The control lines were assigned an arbitrary value of 1, with the respective RL variants calculated as +fold increase or -fold decrease. Due to IP protection, Sample A* and Sample B*, genes whose assays are in the process of being patented, cannot be listed under their actual names in this report.
Figure 4. Graphs Showing Expression of Genes of Interest in RL variants compared to their Controls as measured by qRT-PCR (y axis = fold change)
GAPDH was taken as endogenous control.
HCC1954 was done on 2 replicates.
SKBR3 was done on 2 replicates but 1 replicate gave very high Ct values for GAPDH so only 1 replicate was used.
BT474 was done on 1 replicate.
Discussion and Conclusions
The migration and invasion analysis for SKBR3 was as expected, with its RL variant showing greater propensity for migration (84.4% increase) and invasion (25.7% increase) compared to SKBR3 parent cells. The HCC1954 cell line pairs were seeded too densely to note any differences and should be repeated with fewer cells seeded initially. The increase in migration and invasion could mimic increased mobility/motility and ability to metastasise, respectively, in vivo.
The amount of RNA in each sample cell line was quantified using a NanoDrop Spectrophotometer. The OD260/OD280 ratio ranged from 2.03 to 2.16 and was within the expected range for a pure sample. The OD260/230 range (0.34 to 1.59) was lower than expected (2.0 to 2.2) for pure nucleic acid.
As outlined above, the levels of expression of a number of genes were measured in SKBR3, HCC1954, and BT474 Lapatinib--resistant variants compared to their sensitive parent/control cell lines. The genes analysed were AXL, EGFR, ERBB3, FOXO3, IGF1R, PTEN, Sample A* and Sample B* (*Sample A and Sample B genes and their assays are in the process of being patented, so their actual names cannot be listed in this report for IP protection.) This was to determine if these gene transcripts were up- or down- regulated in the RL cell lines. Expression of EGFR, AXL and Sample B* were found to be most affected by acquiring resistance. As Jones et al (2006) reported, amplification of EGFR and HER2 is frequently observed in human cancers and, as the lines studied in this project are HER2-positive cell lines, the elevated EGFR levels (579x in SKBR3 RL and 201,441x in BT474 RL) may support this finding. AXL is reported to have higher expression levels in more invasive/metastatic cell lines. (Chan et al 2001), and there was a 302x increase in BT474 RL and a 53.40x decrease in SKBR3 RL. Sample B is still being investigated and there is little literature available for the gene.
While confirmatory testing and further analysis is required, the studies performed here suggest that at least three of the genes assessed may have a role in acquired resistance to Lapatinib in HER2-positive breast cancer. More extensive studies of these genes are now warranted to investigate their potential relevance in this setting.