Læknablaðið : fylgirit - 05.01.2015, Qupperneq 55

X V I I V Í S I N D A R Á Ð S T E F N A H Í
F Y L G I R I T 8 2
LÆKNAblaðið/Fylgirit 82 2015/101 55
E 152 miR200c-141 and ∆Np63 are required for breast epithelial
differentiation and branching morphogenesis
Bylgja Hilmarsdóttir1,2, Valgarður Sigurðsson1, Sigríður Rut Franzdóttir1, Markus
Ringnér4, Ari Jón Arason1,2, Jón Þór Bergþórsson1,2, Magnús K. Magnússon1,2,3,
Þórarinn Guðjónsson1,2
1Biomedical Center, Faculty of Medicine, University of Iceland, 2Department of Laboratory
Hematology, Landspítali University Hospital, 3Department of Medical Pharmacology and
Toxicology, Faculty of Medicine, University of Iceland, 4Division of Oncology and Pathology,
Department of Clinical Sciences Lund, Lund University
byh1@hi.is
Introduction: The epithelial compartment of the breast contains two
lineages, the luminal- and the myoepithelial cells. D492 is a breast
epithelial cell line with stem cell properties that forms branching
epithelial structures in 3D culture with both luminal- and myoepihelial
differentiation. We have recently shown that D492 undergo epithelial to
mesenchymal transition (EMT) when co-cultured with endothelial cells.
This 3D co-culture model allows critical analysis of breast epithelial
lineage development and EMT.
Methods and data: In this study, we compared the microRNA (miR)
expression profiles for D492 and its mesenchymal-derivative D492M.
Results: Suppression of the miR-200 family in D492M was among
the most profound changes observed. Exogenous expression of
miR-200c-141 in D492M reversed the EMT phenotype resulting in gain
of luminal- but not myoepithelial differentiation. In contrast, forced
expression of ∆Np63 in D492M restored the myoepithelial phenotype
only. Co-expression of miR-200c-141 and ∆Np63 in D492M restored the
branching morphogenesis in 3D culture underlining the requirement for
both luminal and myoepithelial elements for obtaining full branching
morphogenesis in breast epithelium. Introduction of a miR-200c-141
construct in both D492 and D492M resulted in resistance to endothelial
induced EMT.
Conclusions: In conclusion, our data suggests that expression of
miR-200c-141 and ∆Np63 in D492M can reverse EMT resulting in
luminal- and myoepithelial differentiation, respectively, demonstrating
the importance of these molecules in epithelial integrity in the human
breast.
E 153 Sub-cellular localization of the MITF transcription factor in
melanoma cells
Sigurður Rúnar Guðmundsson, Indriði Einar Reynisson, Alexander Schepsky,
Margrét Helga Ögmundsdóttir, Eiríkur Steingrímsson
Department of Biochemistry and Molecular Biology, Biomedical Center, Faculty of Medicine,
University of Iceland
srg2@hi.is
Introduction: MITF (Microphthalmia-associated transcription factor) re-
gulates development and differentiation of melanocytes and is a key
component in formation of melanoma. Little is known about how sub-
cellular localization of MITF is regulated in melanocytes and melanoma
cells. In this project we characterize which domains of the MITF protein
are involved in nuclear localization and which signaling pathways are
involved.
Methods and data: We generated MITF-EGFP fusion constructs carry-
ing wild type and mutant versions of MITF and transfected into human
501mel (melanoma) and HEK293T (embryonic kidney) cells to map
which domains of MITF are involved in nuclear localization. The sub-
cellular location of MITF was determined using a confocal microscope.
Results: We have observed that a monopartite nuclear localization
signal is located between a.a. 214 and 217 and is required for the nuclear
localization of MITF. Neither DNA binding nor dimerization are necess-
ary for nuclear retention of MITF. We also found that a cytoplasmic
mutant form of MITF colocalizes with LC3, an autophagosome marker.
This might unravel an important degradation pathway affecting MITF
stability.
Conclusions: We have found that four basic residues of the DNA bind-
ing domain are important for nuclear localization. In addition, we have
shown that the carboxyl-end of MITF affects subcellular localization in
unexpected ways, possibly leading to insights into protein turnover.
E 154 The MITF transcription factor network in Melanoma and
Melanocytes
Dilixiati Remina, Erna Magnúsdóttir, Eiríkur Steingrímsson
Department of Biochemistry and Molecular Biology, Biomedical Center, Faculty of Medicine,
University of Iceland
dir2@hi.is
Introduction: The master regulator protein MITF (microphthalmia
associated transcription factor) is essential for melanocyte and mel-
anoma development. In melanoma MITF acts as a molecular switch that
determines whether melanoma cells differentiate, proliferate or become
quiescent migratory cells able to form metastasis. Recently, our labora-
tory has shown that MITF interacts with different transcription factors to
mediate its effects in melanocytes and melanoma. The aim of this project
is to characterize this network with focus on IRF4 and TFEB in order
to determine how these transcription factors mediate both normal and
malignant development in the melanocyte lineage.
Methods and data: We performed chromatin Immunoprecipitation
(ChIP) experiments with 501mel human melanoma cells with an
antibody against MITF followed by quantitative PCR (qPCR) to quanti-
tatively evaluate binding site enrichment. qPCR enrichments of 5 fold
or greater compared to negative control regions are considered bound
by MITF.
Results: We performed ChIP-qPCR on (ChIP) with antibodies that target
MITF, IRF4, in the human melanoma cell line 501mel. The qPCR on the
MITF ChIP reaction showed 10 to 100-fold enrichment for 8 MITF targets
compared to negative two control regions whereas other 6 potential
targets didn’t show good enrichment.
Conclusions: Chromatin Immunoprecipitation coupled with qPCR allo-
wed us to quantify and identify novel MITF binding sites in melanoma.
However some potential targets didn’t show enrichment maybe due to
the effect that MITF binding is depend on MITF protein level.
E 155 The transcription factor MITF regulates autophagy in
melanoma cells
Margrét H. Ögmundsdóttir, Katrín Möller, Sólveig H. Brynjólfsdóttir, Margrét
Bessadóttir, Kimberley Anderson, Eiríkur Steingrímsson
Biochemistry and Molecular Biology, Biomedical Center, Faculty of Medicine, University of
Iceland
kam10@hi.is
Introduction: Autophagy is a process that removes reactive oxygen
species and other substrates that can harm the cell and it‘s DNA. The
transcription factors TFEB and TFE3 control autophagy in normal cells
by regulating genes required for this process. Under normal conditions
these transcription factors are kept in the cytoplasm by the nutrient sen-
sor mTORC1 but when cells are starved they translocate to the nucleus