| Abstract |
Breast cancer, the most common cancer in women, is a heterogeneous disease.
Ηeterogeneity sources are the cell of origin, driver mutations combination, clonal and
epigenetic evolution, which is reflected in the diversity of its phenotypes, therapy response
and clinical outcomes. Those factors affect cell proliferation rates , the frequency and site of
metastases and therapy resistance. The heterogeneity, which results from genetic and
epigenetic changes, may also develop over time within the same subtype or even the same
tumor itself, thus making targeted-personalized approaches a high priority to achieve
effective theparies. To this end understanding the molecular mechanisms that govern the
tumor biological and clinical behavior is of outmost importance. Promyelocytic leukemia
protein (PML) is the core organizer of nuclear structures that facilitate diverse proteinprotein inteactions and posttranslational modifications of key regulatory factors that
mediate multiple effects in cell survival. PML has first recognized as a fusion with the RARa
receptor that causes Acute Myelocytic Leukemia. Following studies assign to PML a tumor
suppressor properties mediated by pro-apoptotic, pro-aging and cell cycle inhibitory signals
in line with lack of expression in samples of primary tumors, including breast cancer.
However, more recent studies, revealed that in a context dependent way, PML may also
show pro-oncogenic properties. Specifically, in chronic myeloid leukemia (CML), in
glioblastomas (GBM) and in some cases of triple-negative breast cancer (TNBC), PML is higly
expressed, helping to maintain the cancer population, either by regulating their cell cycle, or
by contributing to the regulation of cancer stem cells self-renewal. Consequently, PML has a
dual role in tumourigenesis acting as a tumour suppressor or promoter in cell specific
contexts. The aim of this dissertation was to clarify the molecular targets through which PML
regulates the stemness and metastasis of breast cancer cells as a first step that towards
understanding the role of molecular players that determine its pro- or anti-oncogenic action,
using a PML knock down in well characterized human cancer cell models. Our experimental
results suggest that PML loss enhances cell mobility and the mesenchymal features in cell
culture and the aggressive behavior of MDA-MB--231 cell type manifested high graft-site
tumor growth, lymph node and lung metastasis rate but not the epithelial ER positive MCF7
cells. Histology results indirectly point to higher vascularization of PML -KD tumors an affect
that may be mediated by a higher hypoxic response. More importantly, cell lines derived
proangiogenic properties that may result from epigenetic or extensive clonal selection in
vivo in the absence of PML. PML interacts with p53 target factors, such as EMT factors and
HIF1a, regulating their action according to the genetic background of the cells.
Interestingly, we found that various the bHLH type factors that include the HIF1a master
hypoxia regulator and EMT mediators such as Twist and Snail interact with PML and are
likely inhibited from exerting their EMT via DNA binding. Thus PML impedes the aggresive
EMT-metastatic behavior of MDA- MB-231 but not MCF7 cells that do not express EMT
factors or properties. In agreement with the above, transcriptional profiling of these cells
reveals that important biological functions such as cell adhesion, cell cycle, and signaling
pathways associated with cell metastasis are deregulated by PML knock down in MDA–MB231 but not MCF7 cells.
A common link in the above results may the p53 tumor suppressor protein. Wild type
p53 is activated and bound by PML under stress to mediate pro-senescence and apoptotic
functions. We show here that PML binds much stronger to mutant forms of p53 as found in
the MDA-MB-231 (R280K) relative to wild type p53 carried by MCF7 cells. Since mutant p53
is a strong gain of fuction oncogenic driver, we assume that PML is a functional barrier to the
tumor–promoting action of mutant p53 as indicated by TCGA survival data that correlate
high PML expession with better prognosis in mutant p53 but not wild type p53 TNBC
patients. PML loss may thus release activity of mutant p53 to promote metastasis via EMT,
angiogenesis or secretion of exosomal vesicles. Experiments to directly address the role of
the PML in mutant p53 in the above processes are under way.
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