Cancer Research in Switzerland - Krebsliga Schweiz

Irminger-Finger Irmgard | BRCA1-associated protein,
BARD1, a molecular target for breast cancer screening
and cancer therapy (KLS 01962-10-2006)
Breast cancer is the most frequently diagnosed cancer in
the Western world, and the numbers are increasing in the
emerging economies, such as China, India and Brazil. It is
assumed that this latter increase is due to a change in nutritional
lifestyle and might be explained by the increased
consumption of oestrogens through the food chain. Oestrogens
act through the oestrogen receptor alpha (ER-alpha),
which induces the transcription and upregulation of
a variety of target genes, among which are the breast
cancer genes BRCA1 and BRCA1-associated RING domain
protein 1 (BARD1). BARD1 is often described as a protein
binding to BRCA1. It acts as stabilizer of BRCA1 and enhancer
of the E3 ubiquitin ligase activity of BRCA1, which
is important for controlled turnover of many target proteins,
and one of these is ER-alpha. However, BARD1 isoforms
that lack the BRCA1-interaction domain are upregulated
in breast and ovarian cancer, and their expression
is correlated with poor prognostic factors, such as tumour
size, stage and grade. Thus BARD1 isoforms are biomarkers
of cancer progression.
The overall goal of our project was: 1) to characterize the
oncogenic functions of breast cancer-specific BARD1 isoforms
and generate tools for their inhibition; and 2) to
develop methods for their detection in the blood. To reach
these goals we cloned individual isoforms in expression
vectors, expressed them in breast cancer cell lines and
generated antibodies against epitopes specifically expressed
on BARD1 isoforms. With this approach we could
demonstrate that BARD1 isoform delta acts antagonistically
to the function of the BRCA1-BARD1 E3 ubiquitin ligase
in the controlled degradation of the ER-alpha. We
also demonstrated that overexpression of BARD1-delta
leads to ER-alpha accumulation, as it is observed by repression
of full length BARD1 or BRCA1. Importantly, ERalpha
induces expression of BARD1 but also BARD1 isoforms
when activated by oestrogen, which leads to a
feedback loop of increased expression of isoforms. Since
oestrogen is the biggest risk factor for breast cancer, inhibition
of ER-alpha upregulation, based on the inhibition of
BARD1 delta, could be an important novel tool for targeted
cancer therapy.
We also showed that expression of isoform beta antagonizes
the degradation of the mitotic kinase Aurora B, while
the turnover of Aurora B is normally regulated by BRCA1-
BARD1 E3 ubiquitin ligase. Inhibition of Aurora B expression
or activity is important for controlling the proliferation
of tumour cells. Repression of BARD1-beta by small
interfering RNA (siRNA) leads to growth arrest in several
cancer cell lines tested in vitro. Thus, inhibiting BARD1beta
isoform expression can be exploited for the development
of a novel targeted cancer therapy.
To demonstrate that BARD1 isoforms could be detected in
the blood, we used sera from breast cancer patients and
performed enzyme-linked immunosorbent assay (ELISA)
using antibodies specifically recognizing BARD1 isoforms
to detect them in sera from patients with breast cancer.
Further work will be necessary to clearly prove whether
BARD1 isoform detection in the patient’s blood can be
used as diagnostic tool.
Project coordinator
Dr Irmgard Irminger-Finger
Laboratoire de gynécologie-obstétrique moléculaire
Département de gynécologie et d’obstétrique
Maternité
Hôpitaux universitaires de Genève (HUG)
Boulevard de la Cluse 30
CH-1211 Genève
Phone +41 (0)22 382 43 27
irmgard.irminger@unige.ch
Kalberer Christian P. | Role of NKG2D receptor-ligand
interactions in the recognition of human B-cell neoplasms
by natural killer cells (OCS-01870-02-2006)
Human natural killer (NK) cells are important effectors of
the innate immune system and contribute to the first line
of defence against virus-infected cells and tumour cells.
Cognate interactions of activating NK cell receptors and
their ligands result in target cell killing. The activating receptor
NKG2D recognizes ligands that, in humans, belong
to the ULBP and MIC gene families and are crucial determinants
in anti-tumour immunity. Our recent studies in
patients with acute myeloid leukaemia showed that malignant
cells express low levels of ULBP and MIC ligands, resulting
in evasion of leukaemic blasts from immune surveillance
by NK cells. This study addresses the molecular
mechanisms regulating ULBP1 leading to low surface expression
levels in acute leukaemias.
The goal was to investigate the role of the 3’ untranslated
region (3’UTR) in post-transcriptional regulation of ULBP1
expression. Considerable differences in length and sequence
of the 3’UTRs of the ULBP genes suggest that this
region plays a role in differential expression of ULBPs. Indeed,
sequence analysis of 2.4 kb-long ULBP1-3’UTR revealed
potential binding sites for more than 200 micro-
RNAs and the presence of four AU-rich elements (ARE),
the regulatory components of RNA degradation and
translational suppression. Stable or transient delivery of
luciferase reporter constructs containing the full-length
ULBP1-3’UTR sequence with lentiviral vectors or expression
plasmids resulted in a strong reduction of luciferase
activity to 7 – 22 % in Jurkat, HeLa cells and human primary
fibroblasts, indicating a contribution of 3’UTR to the
regulation of ULBP1 gene expression. To determine the
position of regulatory sequences in the ULBP1-3’UTR, we
generated nine vectors carrying different fragments of
ULBP1-3’UTR that all led to significant reductions of luciferase
activity to 19 – 62 %. The suppressive effects were
seen with every fragment along the 3’UTR, suggesting
that the regulatory sequences are distributed over the entire
3’UTR rather than restricted to specific areas. Muta-
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