Oestrogen receptor mutations and their influence on breast cancer growth
Date
2012-03-12
Authors
Amoils, Karin Dagmar
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Abstract
Oestrogen receptor (ER) mutations have been identified for both ERα and ERβ in
previous studies. The effects of the deletion variants due to splice mutations on
clinical parameters, prognosis and treatment were examined in 61 breast
carcinoma patients and 13 control samples from elective reduction mammoplasty
procedures, respectively. RNA extracted from fine needle aspirates (FNAs) of
breast tissue was reverse transcribed and using nested PCR and sequence
analysis the presence of these variants elucidated. Using Χ2 and Fisher’s exact
tests their significance with respect to clinical parameters such as tumour size,
nodal involvement, stage, presence or absence of metastases, menstrual status
and hormone responsiveness was examined. Kaplan-Meier survival analysis was
also determined.
The T-47D breast cancer cell line was cloned with two clones being selected for
further analysis, namely TCA3 (hormone sensitive) and TCC1 (hormone resistant).
These clones were treated for ten passages with oestrogen metabolites, 17-β-
oestradiol and oestriol; oestrogen precursors, androstenedione and cholesterol; an
anti-oestrogen, 4-hydroxy-tamoxifen; and the aromatase inhibitor
aminoglutethimide, respectively. RNA was extracted from the cells initially and
after the tenth passage and the ERα and ERβ exon profiles were examined using
RT-PCR and sequence analysis. After the tenth passage hormone response tests
were performed every 24 hours (up to 96 hours) with cell number being
determined using the MTT assay.
The results indicate that ERα and ERβ variants do not have any affect with respect
to menstrual status and nodal involvement (N). Expression of ERα2 and ERα4 are
required by the mouse monoclonal antibody (DAKO ® Clone 1D5) in the
immunocytochemical assay used for the recognition of the protein in order to
assess ER status and therefore show significance. ERαΔ2 and, contrary to
previous investigations, the variant ERαΔ3 were not found to play a role in
tumourigenesis. ERαΔ5 was observed to be more prevalent in ERα-positive
patients and was usually co-expressed with the complete ERα5 indicating
heterodimerization. ERαΔ5 showed no significance with respect to progression of
disease or response to hormone treatment.
An increase in the ratio of ERαΔ4: wild-type ERα4 indicated an increase in
metastatic potential of diseased tissue. ERα4 and ERαΔ4 heterodimers were
present in both T-47D clones and after 10 passages the TCA3 clone grown in
10-8M aminoglutethimide indicated a complete loss of ERα4 without altering
hormone responsiveness. These results suggest that ERαΔ4 may play a role in
progression of disease but not in the acquisition of tamoxifen resistance.
ERαΔ6 was observed in 15% of patients but not in the T-47D clones or the control
samples. An increase in the expression of ERαΔ6 among patient samples
significantly increased their metastatic potential (p=0.018). ERαΔ6 was also
observed as significant with respect to stage of disease (p=0.023) indicating the
possible relevance of ERαΔ6 in progression of the disease.
ERαΔ7 was the most frequently observed variant and did not show any
significance with regard to any of the clinical parameters examined. The presence
of ERαΔ7 did not show significance with regard to hormone response in vivo but in
vitro the presence of this variant, expressed as a heterodimer with the wild-type
ERα7, conferred greater sensitivity to tamoxifen in the tamoxifen resistant clone
TCC1.
Multiple exon deletions of ERα were also observed. The two more significant
multiple deletion variants were those involving ERαΔ4, namely, ERαΔ2-ERαΔ6
and ERαΔ4-ERαΔ6. The multiple variant ERαΔ4-ERαΔ6 may be involved in
tumour progression.
ERβ variants were not examined in as much detail as ERα variants due to
insufficient material available for analysis. The two domains, the DNA binding
domain and the ligand binding domain, of ERβ were analyzed in a few of the
patients and in the T-47D clones. They were not found to be significant with
respect to the clinical parameters investigated and the ERβ profiles of the TCA3
and TCC1 clones remained unchanged after 10 passages under varying growth conditions.
Description
Ph.D., Faculty of Health Sciences, University of the Witwatersrand, 2011
Keywords
oestrogen, breast cancer