Cancer Research in Switzerland - Krebsliga Schweiz

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Methods and experimental approaches
1) In silico approaches were used to examine RNA expression
levels of PN­1 in multiple primary breast tumours, to
correlate PN­1 expression levels with clinical parameters,
and to investigate the possibility that high PN­1 levels
might have prognostic value in breast cancer. 2) Biochemical
approaches were carried out with different ex vivo
cultured cell lines to study the signalling pathways stimulated
by treatment of cells with purified PN­1 protein. 3)
In vivo tumour studies were performed to examine the effects
of PN­1 ablation on the ability of a metastatic breast
cancer model to form primary mammary tumours and to
form lung metastases.
Results
In our work we used computational analyses to show that
levels of the serine protease inhibitor PN­1 are significantly
higher in high grade compared to low grade breast
cancer. Using breast cancer models with low and high
PN­1 levels we showed that PN­1 treatment of mammary
tumour cells not expressing PN­1 stimulated ERK activity
and matrix metalloproteinase­9 (MMP­9) production via
low­density lipoprotein receptor­related 1 (LRP­1) binding.
Moreover, shRNA mediated ablation of PN­1 expression
in PN­1 overexpressing mammary cancer cells had a
strong negative impact on the ability of the cells to form
lung metastases. Thus, our work uncovered a novel pathway,
whereby the serine protease inhibitor PN­1, via
LRP­1 binding LRP1, activates signalling pathways that
stimulate MMP­9 upregulation and metastatic spread.
Importantly, an analysis of 126 patients with breast cancer
revealed that those whose breast tumours had elevated
PN­1 levels had a significantly higher probability to
develop lung metastases but not metastases to other
sites, on relapse. These results suggest that PN­1 might
become a prognostic marker in breast cancer.
Project coordinator
Prof. Dr. Nancy Hynes
Friedrich Miescher Institut für
biomedizinische Forschung (FMI)
Maulbeerstrasse 66
CH­4058 Basel
Phone +41 (0)61 697 81 07
Fax +41 (0)61 697 39 76
nancy.hynes@fmi.ch
Janscak Pavel | Study of the role of the human
mismatch repair system in telomere metabolism
(OCS 01730­08­2005)
Telomeres are regions of repetitive DNA sequence at the
ends of eukaryotic chromosomes that protect chromosome
ends from being recognized as deleterious DNA
double­strand breaks. In normal cells, telomeres get progressively
shortened due to the inability of DNA polymerases
to fully replicate DNA ends. Critically short telomeres
generate chromosome end fusions that cause loss of replicative
capacity, leading to senescence or apoptosis.
Therefore, it is believed that telomere shortening is a determinant
of cellular life span and acts as a tumour suppressor
mechanism. Cancer cells escape from this type
of control of cellular proliferation by activating a telomere
length maintenance mechanism. A substantial number
of cancers employ a recombination­based mechanism referred
to as alternative lengthening of telomeres (ALT). In
yeast cells lacking telomerase, a reverse transcriptase that
can synthesize telomeric DNA, inactivation of the proteins
involved in the initiation of mismatch repair (MMR), such
as Msh2 and Mlh1, promotes cellular proliferation in a
manner dependent on homologous recombination.
The aim of this project was to explore the role of the MMR
system in telomere metabolism in human cells.
The project utilized different molecular and cell biology
methods in combination with biochemical techniques. Using
various human cell lines, we examined whether the
MMR proteins are localized at telomeres and whether depletion
of these proteins has an effect on telomere integrity.
We found that the MMR proteins MSH2 and MLH1 were
associated with telomeres in ALT­positive cancer cells but
not in ALT­negative cells. We also found that MSH2 and
MLH1 formed a complex with the Werner syndrome helicase/exonuclease
(WRN), which plays a critical role in the
maintenance of telomere integrity. Moreover, our biochemical
experiments with purified proteins indicated
that the MSH2/MSH3 and the MSH2/MSH6 heterodimers
could enhance WRN­mediated unwinding of synthetic
DNA structures that resemble recombination
intermediates. Finally, analysis of recombination events
at telomeres in a human embryonic kidney cell line with
inducible expression of MLH1 revealed an elevated frequency
of telomeric sister chromatid exchanges upon inhibition
of MLH1 expression.
Defects in a subset of MMR genes including MSH2,
MSH3, MSH6, MLH1 and PMS2 are associated with hereditary
non­polyposis colorectal cancer. Our findings
have implications for understanding of the molecular
events underlying the tumourigenic process initiated by
the loss of mismatch repair capacity.
Project coordinator
Dr. Pavel Janscak
Institut für molekulare Krebsforschung
Universität Zürich
Winterthurerstrasse 190
CH­8057 Zürich
Phone +41 (0)44 635 34 70
pjanscak@imcr.uzh.ch