First name
Pietro
Last name
Lo Riso
Year of Study
Research Center
Thesis Title
Induced Pluripotent Stem Cells: An Innovative Tool to Dissect Ovarian Cancer Pathogenesis
Thesis Abstract
Ovarian cancer (OC) has one of the highest death-to-incidence ratios among all
tumor types, which points to the need for novel therapeutic and prognostic
strategies. Indeed, the absence of relevant tumor cell lines that can recapitulate
disease histopathology highlights an acute need for new model systems to study
this pathology. In particular, it is still unclear whether the most common and
aggressive form of this disease, high grade serous ovarian cancer (HGSOC),
could arise from in the ovarian surface epithelium (OSE), as initially thought,
or might be arising from the fimbrial epithelium. Here I addressed these issues
in two complementary ways based on induced pluripotent stem cells: i) the
modeling of Ovarian Cancer by somatic cell reprogramming to pluripotency of
tumor cells; ii) the molecular characterization of HGSOC and its putative cells
of origin. Somatic cell reprogramming, by erasing tumor-associated epigenetic
marks while preserving the underlying genetic mutations, would allow for the
first time the precise dissection of genetic and epigenetic contribution to this
disease, through the differentiation of OC-iPSC into disease-relevant cell types.
I demonstrated the feasibility of OC reprogramming through a non-integrative
platform, showing that OC-derived iPSC are closely similar to human ESC,
and proving their tumoral origin by whole exome sequencing. Moreover, I
showed that independent iPSC clones derived from the same tumor upon
trilineage differentiation in vivo show differential tumorigenic potential. For a
more precise dissection of this phenotype, I set up a differentiation protocol
13
that allows differentiation of pluripotent cells into mesodermal progenitors, that
are precursors of both fimbria and OSE. To isolate a pure population of these
cells, I resorted to CRISPR/Cas9 to integrate a selection cassette in the MIXL1
locus. By this approach, I was able to show correct gene targeting at the
intended site, allowing also for selection of mesodermal progenitors upon
differentiation of normal iPSC. The same approach translated to OC-derived
iPSC would allow to study the effects of genetic mutations deprived of tumorassociated
epigenetic marks during differentiation, both at the stage of
mesodermal progenitors and in cells directed towards the female reproductive
epithelium in vivo. The second approach relies on the identification of specific
molecular features of fimbria and ovarian surface epithelium, the two putative
cells of origin of HGSOC. On this side, I offer a first glimpse on molecular
features of HGSOC cancer and normal gynecological tissues. I could show that
specific DNA methylation signatures of fimbrial epithelial cells and ovarian
surface epithelium cells are partially retained in tumor samples and stratify
HGSOC samples according to the putative cell of origin of this tumor.
Moreover, I show for the first time a description of histone modifications in
primary HGSOC, concentrating on marks of activation/repression sitting on
promoter regions (H3K4me3 and H3K27me3, respectively) and marks that
characterize active/closed-poised enhancers (H3K4me1, H3K27ac and
H3K27me3).
tumor types, which points to the need for novel therapeutic and prognostic
strategies. Indeed, the absence of relevant tumor cell lines that can recapitulate
disease histopathology highlights an acute need for new model systems to study
this pathology. In particular, it is still unclear whether the most common and
aggressive form of this disease, high grade serous ovarian cancer (HGSOC),
could arise from in the ovarian surface epithelium (OSE), as initially thought,
or might be arising from the fimbrial epithelium. Here I addressed these issues
in two complementary ways based on induced pluripotent stem cells: i) the
modeling of Ovarian Cancer by somatic cell reprogramming to pluripotency of
tumor cells; ii) the molecular characterization of HGSOC and its putative cells
of origin. Somatic cell reprogramming, by erasing tumor-associated epigenetic
marks while preserving the underlying genetic mutations, would allow for the
first time the precise dissection of genetic and epigenetic contribution to this
disease, through the differentiation of OC-iPSC into disease-relevant cell types.
I demonstrated the feasibility of OC reprogramming through a non-integrative
platform, showing that OC-derived iPSC are closely similar to human ESC,
and proving their tumoral origin by whole exome sequencing. Moreover, I
showed that independent iPSC clones derived from the same tumor upon
trilineage differentiation in vivo show differential tumorigenic potential. For a
more precise dissection of this phenotype, I set up a differentiation protocol
13
that allows differentiation of pluripotent cells into mesodermal progenitors, that
are precursors of both fimbria and OSE. To isolate a pure population of these
cells, I resorted to CRISPR/Cas9 to integrate a selection cassette in the MIXL1
locus. By this approach, I was able to show correct gene targeting at the
intended site, allowing also for selection of mesodermal progenitors upon
differentiation of normal iPSC. The same approach translated to OC-derived
iPSC would allow to study the effects of genetic mutations deprived of tumorassociated
epigenetic marks during differentiation, both at the stage of
mesodermal progenitors and in cells directed towards the female reproductive
epithelium in vivo. The second approach relies on the identification of specific
molecular features of fimbria and ovarian surface epithelium, the two putative
cells of origin of HGSOC. On this side, I offer a first glimpse on molecular
features of HGSOC cancer and normal gynecological tissues. I could show that
specific DNA methylation signatures of fimbrial epithelial cells and ovarian
surface epithelium cells are partially retained in tumor samples and stratify
HGSOC samples according to the putative cell of origin of this tumor.
Moreover, I show for the first time a description of histone modifications in
primary HGSOC, concentrating on marks of activation/repression sitting on
promoter regions (H3K4me3 and H3K27me3, respectively) and marks that
characterize active/closed-poised enhancers (H3K4me1, H3K27ac and
H3K27me3).
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