Edinburgh Research Archive >
Molecular, Genetic and Population Health Sciences, School of >
Molecular, Genetic and Population Health Sciences thesis and dissertation collection >
Please use this identifier to cite or link to this item:
|Title: ||Role of intracellular signalling pathways in conferring resistance to endocrine therapies in breast cancer|
|Authors: ||Cerqueira, Vera|
|Supervisor(s): ||Langdon, Simon|
|Issue Date: ||2010|
|Publisher: ||The University of Edinburgh|
|Abstract: ||Breast cancer is the most prevalent form of cancer in women and accounts for 519,000
annual deaths (WHO Statistics). It has long been established that oestrogen (E2) stimulates
tumour growth of oestrogen receptor (ER) positive breast cancer and is involved in the
pathogenesis of the disease. Consequently, therapeutic approaches targeting the ER were
developed. The use of endocrine therapy is an integral component in treating breast cancer
however resistance to such drugs is a major limitation. Unfortunately, even initially
responding tumours eventually develop resistance - acquired resistance.
The aim of this study was to determine which intracellular pathways may be important in
conferring acquired endocrine resistance. In order to do so, a three-stage MCF-7 cell model
emulating the clinical development of acquired endocrine was used. MCF-7/LCC1 (LCC1)
and MCF-7/LCC9 (LCC9) cells lines were derived from the oestrogen dependent and antioestrogen
sensitive MCF-7 cell line. LCC1 cells remain responsive to endocrine therapies
but their growth is not dependent on oestrogenic stimulus. LCC9 cells, on the other hand
are fully resistant to endocrine therapies and completely oestrogen independent.
A number of different cell membrane receptors and intracellular pathways have been
implicated in endocrine resistance including HER receptor family, PI3K/Akt & MEK/ERK
pathways. These pathways are of particular interest since they are able to activate ER in the
absence of oestrogenic stimulus. It is likely that several pathways may be important in
conferring resistance to endocrine therapies therefore the experiments in this study
focussed on the transcriptional regulation of HER receptors, the activation of the Akt
pathway and its implication to basic cellular processes.
Following E2 treatment (48h), HER2/3/4 mRNA and protein levels were reduced in MCF-
7 and LCC1 but not in the endocrine-resistant LCC9 cell line as measured by QRT-PCR
and Western blotting. The anti-estrogen fulvestrant (ICI 182,780) reversed the E2
modulation. A previous study has shown that ER and the HER2 promoter compete for
limiting amounts of SRC-1 in oestrogen-responsive ZR-75-1 cells, causing HER2
repression after E2 stimulation (Newman et al.,Oncogene, 19, 490-7, 2000). ER RNAi
abolished E2 repression of HER2 in MCF-7 and LCC1 cells. Furthermore, LCC9 cells have
reduced SRC-1 recruitment to ER (assessed by ChIP) allowing SRC-1 to bind to the HER2 promoter. SRC-1 RNAi reduced HER2 transcription in MCF7 cells in a manner similar to
E2 whilst it did not restore E2 repression in LCC9 suggesting that the latter cells have
alternative mechanisms regulating HER2 transcription. RNAis against the other two p160
co-activators TIF2 and AIB1 did not restore E2 mediated HER2 repression in LCC9 cells.
The importance of redundancy between p160 co-activators was also determined by
performing double knockouts. SRC-1/TIF2 and TIF2/AIB1 double siRNAs had little effect
on HER2 mRNA levels however SRC-1/AIB1 siRNA restored oestrogen mediated
downregulation of HER2 transcription in LCC9 cells. This data indicates that SRC-1 and
AIB1 co-activators play a role in the transcriptional regulation of HER receptor
particularly in MCF-7 and LCC1 cells. The regulation of this transcriptional mechanism is
altered in resistant LCC9 cells but, as evidenced by the double knockouts, p160 coactivators
are still able to affect HER expression in these cells. This mechanism was
further studied in primary breast cancer tumour material.
The importance of the Akt pathway in this cell line model was also investigated as
phospho-Akt levels are elevated in LCC1 and LCC9 cells. This in turn was shown to
activate mTOR and ER (Ser167 residue phosphorylation) thereby contributing to increased
growth and ligand independent activation of the oestrogen receptor respectively.
Activation of PI3K and PTEN is unchanged in LCC1 and LCC9 cells suggesting that these
proteins are not responsible for elevated Akt phosphorylation. In contrast, these cells do
express higher levels of phospho-IGFR due to the high availability of receptor ligands
(IGFI & IGFII). This is likely to be, at least partially, responsible for the elevated Akt
activation. Moreover, the role of Akt isoforms was also determined as they are known to
have different functions. The levels of Akt 2 phosphorylation are higher in endocrine
resistant cell lines in comparison to parental MCF-7 cells. Interestingly, the Akt 3
phosphorylation is present in all cell lines whilst Akt 1 phosphorylation is minimal.
Nevertheless, Akt RNAi studies reveal that Akt 1 and 2 siRNA dramatically reduce growth
in MCF-7, LCC1 and LCC9 cells. These results suggest that Akt 2 phosphorylation may
play a part in conferring endocrine resistance but the other isoforms are also important for
normal cellular growth.
The cell cycle profiles of LCC1 and LCC9 are very similar to MCF-7. Similarly, migration
levels are unchanged in endocrine resistant cell lines. However, in the presence of antioestrogenic
drugs, apoptosis in LCC1 and LCC9 cells in reduced in comparison to the parental MCF-7 cell line. Furthermore, LCC1 and LCC9 cells have higher invasion rates.
The deregulation of HER receptor expression and elevated Akt activation may together
confer survival advantage in LCC1 and LCC9 cells whilst also increasing their invading
|Keywords: ||breast cancer|
|Appears in Collections:||Molecular, Genetic and Population Health Sciences thesis and dissertation collection|
Items in ERA are protected by copyright, with all rights reserved, unless otherwise indicated.