|dc.description.abstract||One group of chemotherapeutics that are used successfully to treat breast cancer, alone or in
combination with other agents, are the taxanes; paclitaxel and docetaxel. They act by interfering
with the spindle microtubule dynamics of the cell causing cell cycle arrest. However, the
complexities underlying the mechanism of action are yet to be fully elucidated. Arguably, one of
the most significant problems with taxanes is chemoresistance. Unfortunately, some patients
are intrinsically resistant to taxanes and others acquire resistance to taxanes as treatment
advances. This problem is exacerbated by a lack of understanding of the mechanisms underlying
Isogenic breast cancer cell lines that were taxane resistant were generated to use as an
experimental model. Paclitaxel resistant (PACR) MDA-MB-231, paclitaxel resistant ZR75-1 and
docetaxel resistant (DOCR) ZR75-1 cell lines were successfully generated by incrementally
increasing taxane dose in respective native cell lines in vitro. An extensive characterisation of
each of the resistant cell lines was conducted, focussing primarily on the 25nM resistant cells
which were determined to be the most clinically relevant dose of taxane. A suboptimal dose of
5nM, a “superoptimal” dose of 50nM and the native, taxane sensitive cells was included.
Dose response cell count experiments were performed that confirmed taxane resistant cells had
been generated. It was shown that MDA-MB-231 native cells were more sensitive to paclitaxel
than the ZR75-1 native cells, suggesting that ZR75-1 cells may already have low level inherent
resistance. The MDA-MB-231 25nM PACR cells were tested to determine whether they retained
PACR when maintained in media containing no paclitaxel. MDA-MB-231 25nM PACR cells were
maintained in a taxane free environment for six months and then rechallenged with taxane.
When rechallenged, the PACR cells previously maintained in the absence of paclitaxel mirrored
the pattern of growth of corresponding PACR cells that had been maintained in the presence of
paclitaxel. This proved that in the absence of paclitaxel, PACR cells did not revert to parent
phenotype. This meant that experiments could be designed to grow cell lines as xenografts in
mice, (in the absence of paclitaxel) & bring in vitro experiments into an in vivo setting. Effects of
taxane treatment on both native and resistant cells were analysed using flow cytometry.
Paclitaxel treatment exerted G2/M block in native MDA-MB-231 cells but when PACR cells were
treated with the same dose of paclitaxel no G2/M block was observed, suggesting that PACR
cells had developed a mechanism for escaping G2/M block. ZR75-1 native lines were also
investigated and we established that treatment with paclitaxel also exerted a G2/M block in
these lines. In future studies this process will be repeated to investigate the effect of taxane
treatment on the ZR75-1 PACR and DOCR lines.
CD 1 nude mice were injected with cells from all five cell lines to grow xenografts, unfortunately
MDA-MB-231 PACR cells failed to grow so they could not be used for further xenograft
experiments. PACR, DOCR and Native ZR75-1 cells did successfully grow as xenografts in mice
and confirmed that all 3 groups showed very similar growth patterns. A cross resistance
experiment was conducted and it was determined that the DOCR xenografts maintained a
taxane resistant phenotype to docetaxel, and not paclitaxel and the PACR xenografts may be
perpetuate the paclitaxel resistant phenotype in xenografts and that there may be cross
resistance to docetaxel in the paclitaxel resistant xenografts. This is the first time that taxane
resistant cell lines grown in this way have been established as xenografts in mice. These cross
resistance experiments represent novel findings and merit further investigation.
Extensive genomic and transcriptomic analyses were carried out on the cell lines to help
identify potential taxane resistance markers. aCGH experiments were carried out to compliment
the illumina experiments. The first set of experiments used DNA from pooled whole female
blood as ref sample and DNA from each of the native and taxane resistant cell lines as test
samples. The second set of experiments used DNA from native cells as a ref sample and DNA
from their respective taxane resistant cells as a test, which allowed areas of loss or gain to be
tracked in the genome as resistance increased. In the MDA-MB-231 cell lines the following areas
of loss extended with increasing resistance: 1p36.13-q44, 6p25.3-q12, 8p, 10p, 19q, X Chr and
the following areas of gain 2p25.3-23.3, 3p24.3-q13.3, 4p16.1-q12, 5q14.3-q31.1, 8q21.13-24.3,
11q15.1-q25, centromeric 12, and centromeric 14. In the ZR75-1 PACR and DOCR cell lines the
areas of loss extended with increasing resistance in the following regions: 7q, 12p and 16q.
For gene expression analysis RNA was extracted from the MDA-MB-231 cell lines, labelled and
hybridised them to illumina human ref 8 vs. 2 chips. Data showed a progressive increase in
mRNA dysregulation as paclitaxel resistance increased. Eleven genes were dysregulated across
all resistance levels in the PACR MDA-MB-231 cells when compared to the relative cell lines;
RGS16, CLDN1, IL7R, P&PP1R14C, COBL, TRPV4, TSPAN8, CD33, NLRP2, P13, and PAGE5. The
experiment was repeated using MDA-MB-231 PACR, ZR75-1 PACR and DOCR cells and resulting
data was analysed to determine genes commonly dysregulated across resistance levels, between
MDA-MB-231 PACR and ZR75-1 PACR and between ZR75-1 PACR and DOCR cell lines. An
extensive literature search was conducted and established four genes of interest in the context
of our genomic and transcriptomic experiments including AURKA, Mdr-1, Stathmin and YY1.
The novel biomarkers identified in the illumina experiments were validated with
complimentary qPCR gene expression experiments looking at expression levels of the eleven
commonly dysregulated genes identified and a panel of 19 other genes with significantly
increased or decreased expression as resistance increased including AURKA, Mdr-1, Stathmin and YY1. Western blots were performed with lysates from the cell lines using a standard panel
of predictive breast cancer markers and AURKA, Mdr-1, Stathmin and YY1. Combining the data
from the genomic study, the gene expression profile, qPCR and Western blotting it was
established that Mdr-1 had increased expression in the taxane resistant ZR75-1 lines and YY1
had increased expression in the MDA-MB-231 PACR line.
Material from the LAPATAX trial was used to observe any transcriptomic changes occurring in
tumours following treatment with docetaxel and to compare them to changes identified in our in
vitro and xenograft models, this allowed the final step to be taken into a translational
environment. LAPATAX (EORTC 10054) is a phase I-II study of Lapatanib and Docetaxel as
neoadjuvant treatment for HER-2 +ve locally advanced/inflammatory or large operable breast
cancer. Tumour material from eighteen core biopsies pre and post treatment was obtained, the
mRNA was extracted, labelled and hybridised to the illumina array. This allowed the changes in
gene expression pre and post docetaxel treatment to be tracked. The gene expression data from
the LAPATAX trial was combined with gene expression data from our cell line panel and
identified two novel putative markers of taxane resistance DUSP1 and FOS. Although sample
size is small this has provided extremely valuable evidence directly from the clinic. These two
novel putative biomarkers are extremely intriguing and certainly merit further investigation,
ideally using additional taxane treated breast tumour tissue.
Ultimately, an isogenic in vitro model of taxane resistance was developed in two different cell
lines and with two different taxanes within one cell line. The cell lines were characterised and
the effect of the taxanes on the cell cycle was determined in the native and taxane resistant lines.
Selected cell lines were grown as xenografts in mice and performed successful cross resistance
studies upon them. A large transcriptomic and genomic analysis was conducted and has
identified a panel of potential taxane resistance markers and areas of loss and gain in the
genome perpetuated by increasing taxane resistance. This analysis was validated using qPCR
and Western blotting. This allowed a panel of novel taxane resistance markers to be identified.
In future studies it is hoped that these targets will be knocked down with shRNA to observe if
the taxane resistant cell lines revert to the parental phenotype. In vitro studies will be
conducted to find agents that may be used to reduce expression of these markers and restore
sensitivity to taxanes and consequently restore the efficacy of these drugs in a clinical setting.
As far as the author is aware this is the first time that isogenic taxane resistant cell lines have
been generated and investigated in this way.||en