Cancer Research in Switzerland - Krebsliga Schweiz

104
Janscak Pavel | Study of the role of mismatch repair
proteins in the cellular response to DNA double-strand
breaks (KLS 02344­02­2009)
Duration: 01.07.2009 – 01.07.2011
DNA damage is a frequent event in the life of a cell. Failure
to repair DNA damage can lead to cell death, and inaccurate
DNA repair can give rise to genomic instability,
which promotes the onset of cancer in mammals. The
most deleterious form of DNA damage is DNA doublestrand
break (DSB). In eukaryotic cells, two mechanistically
distinct pathways are known to efficiently repair
DSBs: non­homologous end joining and homologous recombination.
However, molecular mechanisms underlying
these DNA repair pathways are not completely understood.
Our recent experiments with human cells revealed
that proteins involved in the initiation of post­replicative
mismatch repair (MMR), such as MSH2, MSH3, MSH6
and MLH1, accumulate at sites of chromosomal DSBs generated
by UVA laser. Ongoing research in our laboratory
aims to gain insight into the function of MMR proteins at
the sites of DNA damage in human cells.
The project utilizes various cell biology methods in combination
with laser micro­dissection technology and immunofluorescence
microscopy. Using RNA interference, we
intend to identify the DNA repair factors that are required
for the recruitment of MMR proteins to the sites of DSBs.
DNA repair capacity of cells depleted for the individual
MMR proteins will be assessed using chromosomallybased
reporters for specific DSB repair pathways and by
testing sensitivity of depleted cells to DNA damaging
agents.
Our preliminary data indicate that the MMR proteins accumulate
at DSBs in a manner dependent on MRE11 and
CtIP, which are involved in the initiation homologous recombination.
Germline mutations in the MLH1, MSH2 and MSH6 genes
are the major cause of hereditary non­polyposis colon
cancer. It is anticipated that our study will advance understanding
of the molecular events leading to this most
common form of inherited colon cancer.
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
Krek Wilhelm | Roles of the URI oncoprotein in
B-RAF-signaling and melanoma cancer cell proliferation
(KFS 02690­08­2010)
Duration: 01.02.2011 – 01.02.2013
Malignant melanoma (“black skin cancer”) is one of the
most aggressive cancers. Prognosis for advanced stages is
dismal due to a lack of efficient systemic therapies, with
classical cytotoxic chemotherapy being particularly inefficient.
Elucidating the molecular and cell biological bases of melanomagenesis
is therefore of prime importance and has
already led to findings that have been translated successfully
to the clinics. A central player in melanomagenesis is
BRAF V600E , which is a constitutively active mutant form of
the BRAF kinase, a component of the RAS/RAF/MEK/
ERK­signaling cascade. BRAF V600E is found in about 70 %
of all melanomas, and mouse models demonstrated its
oncogenic potential. Selective BRAF V600E inhibitors have
been developed and their therapeutic efficiency in first
clinical trials is promising. However, after some months
clinical resistance develops, and therefore new therapeutic
strategies are still very much needed. BRAF V600E provokes
overactive RAS/RAF/MEK/ERK­signalling leading
to un con trolled cellular proliferation. Several mechanisms
regulate the activity of this pathway at multiple levels.
Particularly important in the negative regulation of pathway
activity are negative feedback systems mediated by
phosphatases that dampen overactive signalling. That
might also be one of the reasons why the BRAF V600E alone
does not suffice for malignant transformation, but secondary
changes (“second hits”) are a prerequisite.
We recently discovered a novel phosphatase (PP)1­dependent
negative feedback system that acts to restrain
S6K1 survival signalling in response to nutrient and growth
factor stimulation. Moreover, PP1 is a target of oncogenic
alterations that disable PP1­mediated feedback inhibition
on survival signalling in different human cancers. In this
project we are testing whether this PP1­dependent feedback
system also acts on the RAS/RAF/MEK/ERK pathway
and is oncogenically targeted in human melanoma.
In this work we are combining human tissue microarrays
(TMA), quantitative phosphoproteome analyses, RNA
interference and mouse models of melanomagenesis.
From these studies, we expect to discover novel regulatory
mechanisms of oncogenic BRAF V600E signalling and of
RAS/RAF/MEK/ERK­signalling in general.
Project coordinator
Prof. Dr. Wilhelm Krek
Institut für Zellbiologie
ETH Zürich
HPM F42
Schafmattstrasse 18
CH­8093 Zürich
Phone +41 (0)44 633 34 47
Fax +41 (0)44 633 13 57
wilhelm.krek@cell.biol.ethz.ch