1. 1
UNIVERSITA’ DEGLI STUDI DI TORINO
STRUTTURA DIDATTICA SPECIALE DI BIOTECNOLOGIE
TESI DI LAUREA SPECIALISTICA
MORGANA: A KEY PLAYER IN BREAST CANCER
METASTASIZATION
CANDIDATA RELATORE
Elena Busso Prof. Mara Brancaccio
ANNO ACCADEMICO 2015/2015

2. 2
INDEX
INTRODUCTION
MATERIALS AND METHODS
Cell culture
Antibodies, Reagents and Plasmids
Immunofluorescence
Protein extraction and Western Blot
In vivo tumor and metastasis assay
Cell pellet preparation and Immunohistochemical analysis
Migration and Invasion Assay
Gelatin Zymography
RNA isolation and qRT-PCR
Transfection and Luciferase Assay
Statistical Significance
RESULTS
Morgana knockdown cells show impaired migration and invasion in vitro
Morgana knockdown cells show impaired metastasis formation in vivo
Morgana downregulation causes a decrease in MMP-9 activity and expression
Morgana effect on MMP-9 expression is not dependent on ROCK/PTEN/AKT pathway
Morgana regulates MMP-9 expression through the NF-kappaB transcription factor
Morgana overexpressing cells show increased expression of NF-kappaB target genes
Morgana can modulate the NF-kappaB pathway
DISCUSSION AND FUTURE PERSPECTIVES
ACKNOWLEDGMENTS
REFERENCES
FIGURES AND TABLES

3. 3
ABSTRACT
Morgana is a ubiquitously expressed protein, which has been shown to act as an hsp90
co-chaperone and to inhibit ROCK activity. In a small subset of human breast cancer
samples, morgana has been found expressed at higher levels compared to normal
tissues and, moreover, morgana overexpression correlates with tumor grade, mitotic
index and lymph node involvement, regardless of the breast cancer subtype. It has
been recently demonstrated that morgana overexpression in breast cancer cells leads
to chemoresistance, through the ROCK/PTEN/AKT axis. Here we show that morgana is
required for invasion and migration of aggressive breast cancer cells in vitro and for
their ability to form metastasis in vivo. From a mechanistic point of view, we
demonstrated that high morgana levels cause an increase in the transcriptional activity
of NF-kappaB, which in turn drives the transcription of several target genes, including
metalloproteinases and inflammation genes, which have been shown to promote
metastasis formation.

4. 4
INTRODUCTION
Morgana/chp-1 has been characterized as a ubiquitously expressed CHP (CHORD
containing protein), composed of two CHORD domains and a CS (after CHORD-
containing proteins and Sgt1) domain [1]. CHORDs (cysteine and histidine rich
domains) are 60-amino acids Zn2+
binding motifs, characterized by unique cysteine and
histidine sequence patterns [1]. These domains were first identified in the plant
protein Rar1, which is composed of two tandemly repeated CHORD domains and an
additional 20 aminoacid sequence motif, absent in non-plant species [1]. Not
vertebrates, with the exception of yeast, express CHPs with an additional C-terminal CS
domain. The CS domain is also present in Sgt1, a multifunctional protein present both
in plants and animals. While in plants the CHORD and CS domains are present on
distinct proteins (Rar1 and Sgt1) but their physical interaction is required for their
biological function, not vertebrates and vertebrates express CHPs containing both
CHORD domains and a CS domain. Moreover, in vertebrates there are two different
CHP genes, codifying for the ubiquitously expressed morgana and the muscle-specific
melusin, thus suggesting that during evolution the ancestral gene underwent
duplication [2].
It has been demonstrated that morgana/chp-1 knockout mice are embryonic lethal
and that morgana null Drosophila die as instar larvae due to strong proliferation
defects. Moreover, larvae neuroblasts show a strong mitotic phenotype, characterized
by high frequency of diploid cells with supernumerary centrosomes [3]. Interestingly,
the human morgana ortholog can fully rescue the fly centrosome phenotype [3],

5. 5
suggesting a conserved role for morgana between mammals and Drosophila. It has
been shown that morgana can interact with Hsp90 [3], acting as a co-chaperone [4],
and with ROCKI and ROCKII [3], inhibiting their activity. Moreover, mouse embryonic
fibroblasts (MEFs) derived from morgana +/- mice have an higher frequency of
polyploidy cells, supernumerary centrosomes and multipolar spindles [3]. These
characteristics are all important features of cancer progression [5] and, accordingly,
morgana +/- mice are more susceptible to chemical mutagenesis compared to wild-
type cells and display oncogenic features in culture [3]. Importantly, morgana is
strongly downregulated in 67.3% of human breast and 57.7% of human lung cancer
samples compared with control tissues [3], thus suggesting a causative role for
morgana downregulation in cancer onset in human. Furthermore, we recently
demonstrated that morgana haploinsufficient mice develop a myeloproliferative
neoplasm resembling human atypical chronic myeloid leukemia [6]. However, our
tissue array analysis showed that, unexpectedly, morgana is also overexpressed in a
small fraction of human breast (5.4%) and lung cancers (10.3%) [3]. This intriguing
result was further investigated, and recently it has been demonstrated that morgana
overexpression induces transformation in NIH-3T3 cells and strongly protects them
from apoptosis induced by various apoptotic stimuli and from anoikis [7]. From a
mechanistic point of view, high morgana levels inhibit ROCKI, thus destabilizing PTEN
and triggering the PI3K-AKT survival pathway. Accordingly, when morgana is
downregulated in MDA-MB-231 and T47D, PTEN expression increases and leads to cell
sensitization to chemotherapy [7]. In normal breast epithelium-derived cell lines (such
as MCF-10A) and in not aggressive breast cancer cell lines (such as MCF-7), morgana is
expressed at low levels, while breast cancer-derived cell lines (MDA-MB-231, T47D)

6. 6
express morgana at higher levels. Thus, the more aggressive the phenotype, the higher
is morgana expression [7].
Intriguingly, it has been demonstrated that morgana is associated with an
aggressive phenotype in human breast cancers: within different subtypes, morgana
overexpression correlates with high tumor grade, mitosis number and percentage of
Ki67 positive cells [7]. Moreover, morgana is overexpressed with higher incidence
(36%) in triple negative breast cancers, indicating a putative role for morgana in
sustaining a more aggressive phenotype. The term triple negative breast cancer is used
to identify approximately the 15% of breast cancers that lacks the expression of
estrogen receptor (ER) and progesterone receptor (PR) and which do not have the
amplification of the human epidermal growth factor receptor 2 (HER2) [8]. These
cancers are characterized by increased aggressiveness, higher rate of relapse and
worst survival. The absence of a high frequency alteration and the lack of specific
biomarkers is the major obstacle in the development of a successful therapeutic
strategy [8]. Furthermore, morgana correlates with lymph node involvement (pN) [7],
which is an index indicating how much the tumor has spread outside the site of origin,
thus its attitude to give rise to metastasis.
Metastasis are the end product of a multistep cell-biological process called the
“invasion-metastasis cascade”, which involves migration of cancer cells from their site
of origin, dissemination into distant organs and subsequent adaptation to the new
extracellular environment [9]. During the metastatic progression, tumor cells need to
overcome a series of steps: exit their primary sites of growth (local invasion and
intravasation), translocate systemically (survive in the circulation, arrest at distant

7. 7
organ sites, extravasate), and adapt to survive in the foreign microenvironment of
distant tissues (micrometastasis formation and metastatic colonization) [10]. Each step
of this process not only requires cancer cells to acquire several properties by genetic or
epigenetic alteration, but also depends upon the cooperation between cancer and
stromal cells. Alteration of cellular adhesion and cell motility, resistance to
extracellular death signals, disruption of basement membrane and extracellular matrix
(ECM) are among the most common alterations needed for metastasization [9].
In particular, ECM proteases are intrinsically associated with breast tissue
remodeling and cancer, since they mediate enzymatic degradation of the extracellular
matrix and of the basement membrane [11]. The activity of matrix proteases is
normally under tight control through specific localization, autoinhibition and secreted
tissue inhibitors [9]. Cancerous cells use diverse mechanisms to disrupt this tight
regulation and unleash proteolytic activity on the basement membrane and interstitial
extracellular matrices. In addition to facilitating tumor environment, extracellular
proteases can generate a diverse array of active cleaved peptides that can modulate
migration, cancer-cell proliferation, survival, and tumor angiogenesis [12]. Matrix
metalloproteinases are the most prominent proteases during breast cancer
progression: studies have reported that within the matrix metalloproteinase (MMP)
family, gelatinases A (72 kDa gelatinase, type IV collagenase and MMP-2) and B (92
kDa gelatinase, type IV collagenase and MMP-9) play a critical role in ECM degradation
and cell migration, leading to tumor cell invasion in breast cancer [13]. Elevated MMP-
9 levels have been functionally linked to elevated metastasis formation in a number of
tumor types, such as brain, prostate, bladder and breast tumors [12]. Consequently,
inhibiting the expression of MMP-9 and/or its upstream regulatory pathways may

8. 8
prove to be effective in treating malignant tumors, including breast cancer.
Unfortunately, this first effort has been unsuccessful due to the several joint disorders
caused by these inhibitors [14], thus better understanding the activity of these
proteins is crucial in order to design a new generation of more effective protease
inhibitors. MMPs are not coordinately regulated at the transcriptional level: their
transcription is independently regulated, and each cell displays a proteolytic
phenotype in response to a particular stimulus. Several factors have been
demonstrated to be involved in the overexpression of MMPs in tumors, among them
interleukin (IL-1) and tumor necrosis factor (TNFα). The signal transduction pathway
that mediates the expression of metalloproteinases are diverse and the most
important is the mitogen activated kinase (MAPK) pathway, which stimulates or inhibit
MMPs expression depending on the cell type. Most of these pathways converge at the
transcription factors AP-1 and NF-kappaB, whose binding sites are present in the
promoter of MMP-9 [15].
Since metastasization to distant organs is causative for 90% of all breast cancer
deaths [16], shedding some light on the molecular mechanisms at the basis of this
process is clearly crucial. Herein, we propose morgana as an important player in breast
tumor metastasization, supporting cancer cell invasion mainly by activating NF-kappaB
transcription factor, thus promoting the transcription of prometastatic and
inflammation-related genes.

9. 9
MATHERIALS AND METHODS
Cell culture
All human breast cancer cell lines were purchased from the American Type Culture
Collection (ATCC, Manassas, VA, USA) and propagated and maintained according to
protocols supplied by ATCC. MDA-MB-231 cells were cultured in DMEM, 10% FBS, 5%
PS. MCF-7 cells were cultured in MEM with 10% FBS, 5% PS and 1% Insulin. BT-549
cells were cultured in RPMI 5% PS, 10% FBS, 1% Insulin. MCF10A were cultured as
previously described [17]. Morgana knock-down in MDA-MB-231 and BT-549 was
performed by infecting cells with pGIPZ lentiviral particles expressing two different
morgana shRNAs together with the TurboGFP (Open Biosystems). Empty pGIPZ
lentiviral vector, expressing TurboGFP reporter, was used as control. MCF-7 and MCF-
10 overexpressing morgana were obtained using a pLVX lentiviral vector coding for
mouse morgana.
Antibodies, Reagents and Plasmids
Western blotting was performed using the following antibodies: anti-morgana P1/PP0
[3], actin, ROCK I, MLC2, MMP2, P-IkBα (Tyr42/46) (Santa Cruz Biotechnologies),
phospho-MLC2, PTEN, phospho-AKT, AKT, IkBα, P-IkBα (Ser32/36), IKKβ (Cell
Signaling), vinculin and β-tubulin (Sigma-Aldrich), MMP-9, IL-6 (Abcam). Bovine Serum
(FBS), Fetal Calf Serum (FCS), Penicillin-Streptomycin (PS) and Lipofectamine 2000
were purchased from Invitrogen (Invitrogen). Epidermal Growth Factor (EGF), heparin,
insulin and hydrocortisone were from Sigma (Sigma-Aldrich) and TNFα was from Tocris

10. 10
(Tocris Bioscience). Plasmids p1242-3x-KB-L and 3xAP1pGL3 were purchased from
Addgene, pGL2, pGL3 and pRL-TK from PROMEGA.
Immunofluorescence
Cells grown on coverslips were fixed in PFA for 10 minutes at RT. Cells were then
incubated for 1 hour with the following primary antibodies: mouse anti-morgana
P1/PPO [3], rabbit MMP-9 (Abcam). Primary antibodies were detected by 1-hour
incubation with FITC- or RITC-conjugated secondary antibodies (Invitrogen). Nuclei
were counterstained with DAPI (Sigma-Aldrich).
Protein extraction and Western blot analysis
Cells were lysed in Tris-buffered saline (1% Triton X-100, 10 mM NaF, 1 mM PMSF, 1
mM Na3VO4 and protease and phosphatase inhibitors from Roche). To detect the
phosphorylation of MLC2 cells were lysed with SDS buffer (1% SDS, 60 mM Tris HCl pH
6.8). Samples were analyzed by Western blotting and detected by the
chemiluminescent reagent LiteAblot (Euroclone).
In vivo tumor and metastasis assays
The use of animals was in compliance with the Guide for the Care and Use of
Laboratory Animals published by the US National Institutes of Health and was
approved by the Animal Care and Use Committee of University of Torino. 106
MDA-
MB-231 were resuspended in 100 μl of PBS mixed with 100 μl of matrigel and injected
subcutaneously into the left flank of seven-week-old female SCID mice (Charles River
Laboratories, Wilmington, MA). Tumors were measured with a caliper and 4 weeks

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after injection mice were dissected and tumors were weighted. For experimental
metastasis assays, 5x105
MDA-MB-231 cells (in PBS) were injected into the tail vein of
7-week-old female immunocompetent NSG mice (Charles River Laboratories). Mice
were dissected 4 weeks later and macrometastases were counted at Nikon SMZ1000
stereomicroscope. Spontaneous metastases were evaluated in 7-week-old female
immunocompetent NSG mice injected with 1x106
MDA-MB-231 cells (in PBS) and
dissected 8 weeks later. Macrometastases were counted at Nikon SMZ1000
stereomicroscope. For all metastasis studies, organs were formalin fixed, cut in small
pieces and paraffin embedded, sectioned and haematoxylin and eosin (H&E) stained.
Micrometastases were evaluated on specimens, with an Olympus BH2 microscope, on
at least three different sections.
Cell pellet preparation and Immunohistochemistry
Cells were cultured in 150 mm plates until they reach confluence, then they were
trypsinized and resuspended in culture medium. After being transferred in a 15 ml
tube, cells were fixed in 10% formalin over night. The cell pellet was then washed with
ethanol 75% and paraffin embedded. For immunohistochemical analysis sections were
deparaffinized and permeabilized with TBS 0.1% Triton for 10 minutes. Endogenous
peroxidases were inhibited by incubating sections with 6% H2O2 for 10 minutes.
Antigen retrieval was performed at 98°C for 30 minutes in citrate buffer pH 6. The
sections were then saturated with TBS 3% milk for 30 minutes at RT and then
incubated with the primary antibodies (IL6 1:600; anti-morgana P1/PPO 1:200) diluted
in TBS plus ¼ of the final volume of blocking buffer, for 30 minutes at RT. After rinsing
in PBS, EnVision+ System Labelled Polymer-HRP (Dako) anti-mouse were used to reveal

12. 12
staining. The reaction was developed using a solution of 3-3’-diaminobenzidine and
H2O2 for 2 minutes. The nuclei were finally counterstained with Mayer’s haemalum.
Migration and invasion assays
To measure migration 7.5x104
MDA-MB-231 were seeded in serum-free media in the
upper chambers of cell culture inserts (transwells) with 8.0 μm pore size membrane
(24-well format, Becton Dickinson, NJ). Invasion assays for MDA-MB-231 were
performed using BioCoat TM Matrigel Invasion Chambers with 8.0 μm pore size
membrane (Becton Dickinson). For migration and invasion, the lower chambers were
filled with complete growth media. After 24 h, the migrated cells present on the lower
side of the membrane were fixed in 2.5% glutaraldehyde, stained with 0.1% crystal
violet and photographed using an Olympus IX70 microscope. Migration and invasion
were evaluated by measuring the area occupied by migrated cells using the ImageJ
software. For the wound healing assay, MDA-MB-231 cells were cultured in 6-well
plates. When 90% confluent, cells were starved 24 hours. Then, a wound was
scratched in the center of the cell monolayer by a sterile plastic pipette tip. The debris
was removed by washing with PBS. The wound was photographed at time 0 and after
24 hours at Zeiss microscopy (Carl Zeiss). The percentage of wound closure was
calculated using Axio Vision programme.
Gelatin zymography
For gelatin zymography, conditioned media and total protein extracts were collected
from confluent MDA-MB-231 cells maintained in serum-free media for 24 hours and
processed as described [18]

13. 13
RNA isolation and qRT–PCR
Total RNA was isolated from MDA-MB-231, MCF-7 and MCF10A using Trizol Reagent
(Invitrogen Life Technologies), following the manufacturer’s recommendations. RNA
was reverse transcribed by using Applied Biosystem high capacity cDNA reverse
transcription kit. Gene expression analysis was performed using TaqMan Gene
Expression Assays (Applied Biosystems) on an ABI Prism 7900HT sequence detection
system (Applied Biosystems). We used 18s as the endogenous control throughout all
experimental analysis. Analysis was performed using the Δ-ΔCt method to determine
fold changes. We used gene-specific primers and the Universal Probe Library System
(Roche Applied Sciences, Indianapolis, IN).
Transfection and Luciferase Assay
Plasmids for the Luciferase Assay were purchased from Addgene: p1242-3x-KB-L,
containing 3 NF-kappaB binding sites upstream of the Firefly Luciferase gene and
3xAP1pGL3, containing 3 AP-1 binding sites upstream of the Firefly Luciferase gene.
For the Luciferase Assay 80000 cells were plated on a 24-well plate. 24 hours later,
cells were co-transfected using Lipofectamine 2000 (Invitrogen) according to the
manufacturer’s recommendations with 150 ng of pRL-TK Vector (PROMEGA)
containing the Renilla gene, used as a normalizer and internal control, and with 650 ng
of reporter vector (p1242-3x-KB-L or 3xAP1pGL3), or with empty vector pGL2 or pGL3,
respectively (PROMEGA). 24 hours after transfection Dual-Luciferase Reporter Assay
were performed by Glomax instrument (PROMEGA). Results are calculated as fold

14. 14
changes and shown as means of Firefly Luciferase activity normalized on Renilla
luciferase activity.
Statistical significance
The data are presented as means ± s.e.m. In statistical analyses, significance was
tested using a two-tailed Student's t test or, when required, one or two-way ANOVA
with Bonferroni's correction. For all analyses, a minimum value of P<0.05 was
considered significant. All statistical analyses were performed using GraphPad Prism 4
(GraphPad Software version 4.0).

15. 15
RESULTS
Morgana knockdown cells show impaired migration and invasion in vitro
Since we wanted to asses if morgana could play a role in migration and
invasion, we generated MDA-MB-231 cells infected with an empty vector or with two
different shRNA against morgana. We then evaluated the amount of morgana in these
cells by Western Blot and we confirmed that cells transduced with two shRNA showed
a considerable reduction in morgana protein levels (Fig.1A). MDA-MB-231 are highly
metastatic cells, which display a strong ability to migrate and invade. Strikingly,
morgana downregulation in these cells impaired their ability to migrate, assessed both
by wound healing assay and transwell migration assay (Fig. 1B, C). Moreover, we
measured the invasive capacity of MDA-MB-231 infected with shmorgana compared
with control cells, by performing a transwell invasion assay through matrigel.
Interestingly, morgana downregulated MDA-MB-231 displayed a strong reduction of
their invasive capacity, compared to control cells (Fig. 1D). Thus, these results indicate
that morgana downregulation causes an impairment in the migration and invasion
capacity of MDA-MB-231.
Morgana knockdown cells show impaired metastasis formation in vivo
To examine morgana ability to regulate breast cancer cell metastasis formation in vivo,
1x105
MDA-MB-231 cells infected with an empty vector or with a shRNA against
morgana were injected in the tail vein of immuno-deficient NOD/SCID/IL-2Rγc null
(NSG) mice. As expected, control cells efficiently led to macrometastasis formation in
lung, spleen, liver and heart, but no macrometastasis were found in mice receiving

16. 16
cells in which morgana was downregulated (Table 1). We then analyzed the
histological sections of several organs for the presence of micrometastasis: mice
injected with control cells displayed several micrometastasis in all the organs analyzed,
while mice injected with shmorgana MDA-MB-231 displayed few micrometastasis in
the lung (Fig. 2A) but not in other organs. Furthermore, we analyzed by
immunohistochemistry morgana expression in sections of tumors and metastasis
developed by mice inoculated with shmorgana cells, revealing that even the few
micrometastasis developed by these mice presented high morgana levels, comparable
with the one of control cells (Fig. 2B). This evidence suggests that probably these
micrometastasis were induced by escaper cells, which start re-expressing morgana at
high levels again, further supporting the idea that morgana is crucial for
metastasization. To assess morgana ability to promote spontaneous metastasization
from primary tumors, we subcutaneously inoculated 1x106
MDA-MB-231 in the
mammary fat pad of immunodeficient NSG mice. Even in this case, only control cells
gave rise to macrometastasis in the lung (Fig. 2C), thus confirming our previous results.
Moreover, we didn’t find any differences in size and weight of primary tumors (data
not shown), and this is in line with our previous observation that shmorgana cells do
not show an impairment of in vitro proliferation compared to control cells [7], Thus,
morgana role in promoting metastasis formation is to be directly related to an
enhancement of the metastatic ability of cells, and not to proliferation effects.
Morgana downregulation causes a decrease in MMP-9 activity and expression
It is well established that some matrix metalloproteinases such as MMP2 and
MMP-9 play a pivotal role in degrading the extracellular matrix, thus promoting cell

17. 17
invasion. Elevated MMP-9 expression correlates with increased metastatic potential in
a number of tumor types, such as brain, prostate, bladder and breast tumors [12]. For
this reason, we decided to assess the activity and expression of these
metalloproteinases in control and morgana downregulated MDA-MB-231.
Interestingly, shmorgana MDA-MB-231 showed a reduction in the activity of MMP-9,
but not MMP2, both in the intracellular and extracellular compartments, as assessed
by gelatin zymography (Fig. 3A, B). Moreover, we evaluated MMP-9 protein levels,
revealing that morgana downregulated cells have a decreased MMP-9 intracellular and
extracellular protein level, while no significant changes were detectable regarding
MMP2 (Fig. 3C, D). These data have been further confirmed by immunofluorescence
(Fig. 4A, B). In order to assess if morgana is able to control MMP-9 expression not only
at the protein level, but also at the transcriptional level, we performed reverse
transcriptase PCR followed by quantitative Real Time PCR, and we found that morgana
downregulated cells displayed a strong reduction also in MMP-9 mRNA levels (Fig. 4C).
Accordingly, also from the transcriptional point of view MMP2 remains unchanged in
morgana downregulated cells (Fig. 4D), suggesting a specific role for morgana in the
regulation of MMP9 transcription.
Morgana effect on MMP-9 expression is not dependent on ROCK/PTEN/AKT pathway
Our previous works demonstrated that morgana is able to bind and inhibit
ROCKI and II [3] [7] and that through ROCK inhibition morgana downregulates PTEN
expression levels, thus enhancing Akt phosphorylation [7]. Through their action on
cytoskeleton and actinomyocin contractility, Rho kinases play a central role in the
regulation of cell migration and impacts on several components of the metastatic

18. 18
process, including migration, local invasion and cell proliferation [19]. Given that the
PI3K/AKT pathway has been reported to induce cancer cell invasion [20], we tested if
morgana dependent MMP-9 transcription was related to this pathway, by treating
control and shmorgana MDA-MB-231 with the ROCK inhibitor Y27632 and the PTEN
inhibitor vo-ohpic. The activity of the two inhibitors was assessed by Western Blot: as
expected, MDA-MB-231 cells treated with Y27632 showed a decreased
phosphorylation of MLC2, which is a known substrate for ROCK (Fig. 5A), while MDA-
MB-231 treated with vo-hopic showed an increased AKT phosphorylation (Fig. 5B).
Surprisingly, both inhibitors failed to restore normal MMP-9 expression (Fig. 5C, D),
thus indicating that morgana activation of MMP-9 expression is independent on the
ROCK/PTEN/AKT axis.
Morgana regulates MMP-9 expression through NF-kappaB transcription factor
It is known that among the many signals activating MMP-9 expression, most of
them converge on two major transcription factors controlling MMP-9 transcription,
which are AP-1 and NF-kappaB [21] [22]. Therefore, we performed a luciferase assay in
order to understand if morgana could influence the activity of these transcription
factors. We used a NF-κB–responsive reporter plasmid and an AP-1 responsive
reporter plasmid, in which the expression of Firefly luciferase is controlled by a
cassette containing three binding sites for NF-kappaB and AP-1, respectively. We
demonstrated that the activity of AP-1 was unchanged when morgana is
downregulated in MDA-MB-231 (Fig.6A), while on the contrary, the activity of NF-
kappaB significantly decreased in the same cells downregulated for morgana (Fig. 6B).
In order to confirm this result, we generated BT-549 cells infected with an empty

19. 19
vector or with two different shRNA against morgana and we repeated the same
experiment on these cells: again, morgana downregulation could inhibit the activity of
NF-kappaB in these cells (Fig. 6C). In line with these data, NF-kappaB activity increased
when morgana is upregulated in MCF-7 and MCF10A (Fig. 6D, E). Thus, we
demonstrated that morgana is able to regulate NF-kappaB, but not AP-1,
transcriptional activity.
Morgana overexpressing cells show increased expression of NF-kappaB target genes
It is well established that NF-kappaB is a pivotal transcription factor involved in
the regulation of many inflammatory genes such as TNFα, TGFβ, interleukins and
chemokines. Therefore, to further confirm our previous data, we analyzed by qRT-PCR
the expression of some NF-kappaB target genes. As shown in Fig. 7A, shmorgana MDA-
MB-231 cells showed a reduction in MMP-9, IL1α, IL1β, IL6, CCL5 and IL24 mRNA,
compared with control cells. In line with this result, the transcription of some NF-
kappaB dependent genes was upregulated in MCF-7 and in MCF10A cells
overexpressing morgana, upon treatment with TNFα for 4 hours to activate the
transcription of these genes that otherwise is almost completely blocked (Fig. 7B, C).
Eventually, we further confirmed that morgana downregulation can cause a decrease
in IL6 at the protein level: we performed immunohistochemical analysis on cell pellets
of MDA-MB-231 infected with an empty vector of downregulated for morgana, and we
observed that when morgana was downregulated there was a decrease in IL6
production (Fig. 8A). Moreover, we repeated the same experiment on sections of
primary tumors developed by mice injected with MDA-MB-231 infected with an empty
vector or with a shRNA against morgana. As expected, tumors derived from control

20. 20
cells displayed high levels of IL6, while tumors developed by mice injected with cells in
which morgana was downregulated displayed a decrease in the amount of IL6 (Fig. 8B),
confirming the impairment of interleukin 6 production in morgana knockdown cells
also at the protein level.
Morgana modulates NF-kappaB pathway
In order to understand how morgana can affect NF-kappaB activity, we looked at the
protein level of the major player in NF-kappaB pathway. Interestingly, when morgana
is downregulated in MDA-MB-231 there is a decrease in the phosphorylation of IkB on
serine 32 and serine 36 (Fig. 9A). Further confirming this result, morgana
downregulation causes a decrease in the serine phosphorylation of IkB also in BT-549
cells (Fig.9B). Accordingly, when morgana is overexpressed in MCF-7 and in MCF10A,
there is an increase in IkB serine phosphorylation (Fig. 9C, D). To conclude, we
demonstrated that morgana can enhance NF-kappaB activity by increasing the serine
phosphorylation of IkB, but the specific mechanism by which it acts has still to be
elucidated.

21. 21
CONCLUSIONS AND FUTURE PERSPECTIVES
Morgana has been previously characterized as an hsp90 co-chaperone involved
in the control of genomic stability maintenance by regulating the centrosome
duplication via ROCKII kinase. In fact, morgana haploinsufficiency causes multiple
centrosomes, multipolar spindles and increased tumorigenicity induced by chemical
mutagens in mice [3]. Moreover, it has been recently shown that morgana
heterozygous mice develop with age a lethal myeloproliferative disease that resembles
human atypical chronic myeloid leukemia (aCML), preceded by ROCK hyperactivation,
centrosome amplification and aneuploidy [6]. The importance of morgana in
preventing cancer progression is highlighted by the evidence that a consistent fraction
of breast and lung tumors displays low levels of morgana [3]. All these evidences
strongly suggest that morgana acts as a tumor suppressor. However, our tissue array
analysis showed that a small subset of breast and lung tumors overexpresses morgana
[3]. Further analyzing this intriguing result, we demonstrated that morgana
overexpression induces transformation of NIH-3T3 and strongly protects them from
various apoptotic stimuli, such as chemotherapic treatment [7]. In particular, the
mechanism whereby morgana drives cancer progression involves the inhibition of
ROCKI, thus leading to a destabilization of PTEN and a subsequent increase in the
phosphorylation of AKT [7]. Hereby we show a second mechanism with whom
morgana have a pro-tumorigenic role: morgana is able to promote metastasis
formation by activating the NF-kappaB transcription factor. In particular, morgana
overexpressing cells are resistant to anoikis, which is a typical feature of
aggressive/metastatic cancer cells (data not shown). Moreover, morgana

22. 22
downregulation in MDA-MB-231 leads to a consistent decrease in the migration and
invasion of this aggressive cell line. More importantly, MDA-MB-231 downregulated
for morgana lose their ability to give rise to metastasis in NSG mice, both when
subcutaneously and tail vein injected. From a mechanistic point of view, we
demonstrated that morgana downregulated cells possess lower MMP-9 activity, as
assessed by gelatin zymography. MMP-9 is a very well characterized metalloproteinase
that has been associated with metastasis formation in various types of cancers [23].
Moreover, MMP-9 level is reduced both at the protein and at the mRNA levels in
morgana downregulated cells, while no changes are seen in regarding MMP2
metalloproteinase. It has been demonstrated that the PI3K/PTEN/AKT pathway is able
to activate MMP-9 expression, thus favoring metastasis formation in various tumor
types [20]. Nevertheless, morgana dependent activation of MMP-9 expression is not
dependent on this pathway, since nor the ROCK inhibitor (Y27632) nor the PTEN
inhibitor Vo-ohpic are able to revert the phenotype. Instead, interestingly, we
demonstrated that morgana can modulate MMP-9 expression by regulating the NF-
kappaB pathway.
NF-kappaB is a family of transcription factors composed of 5 members: RelA
(also called p65), Rel B, c-Rel, NF-kappaB1 (also called p105) and NF-kappaB2 (also
called p100) [24]. They bind to 9-10 base pair DNA sites with great variability (5’-
GGGRNWYYCC-3’; R = A or G, N = any nucleotide, W = A or T, Y = C or T) and they can
form homodimers or heterodimers with different transcription-modulating properties
[24]. NF-kappaB contributes to the transcription of four main classes of genes: genes
that are involved in negative-feedback control of NF-kappaB itself (such as IKBα, IKBβ,
A20), genes involved in cell proliferation (Cyclin D1, c-Myc), genes that codify for

23. 23
proteins with immunomodulatory functions (chemokines, cytokines, etc.) and anti-
apoptotic genes (BCL-XL, c-FLIP) [25]. Moreover, The NF-kappaB family of transcription
factors plays a pivotal role in both promoting and maintaining an invasive phenotype,
by activating the transcription of genes involved in the epithelial-to-mesenchymal
transition, inflammatory genes and metalloproteinases [26]. In our model, morgana-
dependent NF-kappaB activation leads to increased transcription of several target
genes, including inflammatory genes (IL1α, IL1β, CCL5, IL24, TGFβ, etc.) and
metalloproteinases (MMP-9). Moreover, it should be noticed that NF-kappaB is not
present on MMP2 promoter, thus explaining why we did not see any differences in the
transcription of this metalloproteinase. On the other hand, we did not see any
difference in the transcription of EMT related genes, such as Twist, Snail and Slug (data
not shown). This might be due to the presence of other genetic and epigenetic
mechanisms controlling the transcription of these genes besides morgana, thus
probably morgana alone is not sufficient to induce their transcription is these specific
cellular settings.
There are two main pathways of NF-kappaB activation: the classical pathway
(which concerns RelA, C-Rel and NF-kappaB 1) involves the activation of the IKK
complex (especially IKKβ) which phosphorylates IKB, thus targeting it to ubiquitin-
dependent degradation and subsequent liberation of NF-kappaB dimers that can
translocate to the nucleus and activate the transcription of NF-kappaB-dependent
genes; the alternative activation pathway (which affects NF-kappaB 2 and RelB)
involves activation of IKKα and NIK, inducing phosphorylation-mediated activation of
NF-kappaB 2, that can translocate to the nucleus and activate the transcription [25].
Thus, in the classical activation pathway, IKBα held NF-kappaB in the cytoplasm

24. 24
inhibiting its transcriptional activity, and IKKβ-mediated phosphorylation of IKBα on
two conserved serine residues (S32-S36) leads to its ubiquitination and subsequent
proteasomal degradation [25]. We demonstrated that morgana downregulated cells
have a minor activation of NF-kappaB pathway and, accordingly, morgana
overexpressing cells present an hyperactivation of the NF-kappaB pathway. Moreover,
we found that high morgana levels are combined with higher P-IKBα (on S32/S36).
Another less studied phosphorylation site on IKBα is the tyrosine phosphorylation
(Y42-Y44), which causes NF-kappaB activation without proteolytic degradation of IKBα
[27]. Nevertheless, we show that these alternative sites of phosphorylation on IKBα
are not affected by morgana (data not shown). All things considered, the key evidence
is that morgana overexpression induces an increase, while morgana downregulation
causes an impairment in NF-kappaB transcriptional activity, although the exact
mechanism whereby morgana modulates this pathway and the precise involvement of
IkB degradation is still to be elucidated.
Constitutively activated NF-kappaB transcription factors have been associated
with several aspects of tumorigenesis, including cancer-cell proliferation, apoptosis
prevention, tumor angiogenesis and inflammation, and increased metastatic potential
[28]. In particular, some studies suggests that NF-kappaB activation might be one of
the early events in breast cancer pathogenesis, since it is activated in most human
breast cancer cells, regardless of hormone-dependency status [29]. Moreover, NF-
kappaB activation has been reported to boost metalloproteinase-dependent matrix
destruction by cancer-cells, thus promoting metastasis formation [30]. Among breast
cancer patients the mortality results entirely from invasion and metastasis of cancer
cells in distant organs, and this is true especially for triple-negative breast cancers.

25. 25
Triple negative breast cancers are generally more aggressive than the other subtypes,
with a higher rate of relapse and decreased overall survival in the metastatic disease
[31]. In particular, those patients who achieve pathological complete response to
neoadjuvant chemotherapy have survival rates similar to those of other breast cancer
subtypes. On the other hand, patients who do not respond to standard-of-care
chemotherapy have significantly worse survival and higher rate of relapse within the
first 3 years after treatment [32]. We demonstrated that morgana is expressed with
higher incidence (36%) in triple-negative breast cancer samples, compared with other
subtypes, thus indicating a possible role for morgana in promoting this aggressive
phenotype. Moreover, it is known that NF-kappaB constitutive activation is
characteristic of the basal-like subtype breast-cancer cell lines [33] and this
transcription factor has been associated with increased migration, invasion and
metastatic potential of triple-negative breast cancer cells [34] [21]. Furthermore,
TNBCs are characterized by a high degree of heterogeneity, both inter and intratumor,
and years of studying have failed in finding a single common alteration to be targeted,
thus hindering the development of successful therapeutic strategies [8]. The lack of a
high frequency oncogenic driver is the main cause of the poor outcome of this disease
compared with the other subtypes. Therefore, there is clearly a major need in
understanding the molecular basis of TNBCs and in identifying specific target in order
to develop effective therapeutic strategies. Interestingly, morgana ability to enhance
NF-kappaB transcriptional activity and its enrichment in triple-negative breast cancers
strongly support the idea that morgana is a key player in promoting the aggressive
phenotype of triple-negative breast cancers.

26. 26
It should be noted that morgana is an hsp90 co-chaperone [4]. Hsp90 is a key
component providing maintenance of cellular homeostasis and can interact with two
classes of proteins: more than 200 client proteins whose correct conformation and
activation is promoted by hsp90, and “co-chaperones”, which are accessory proteins
that assist and collaborate with hsp90. Since chaperone proteins are required for
stabilization and activation of numerous client proteins involved in essential cellular
processes like signal transduction pathways, it is possible that morgana modulates the
NF-kappaB pathway through her chaperone activity. Moreover, there are some
evidences showing that hsp90 itself, together with other co-chaperones, is able to
activate the NF-kappaB pathway. In particular, hsp90 and Cdc37, another hsp90 co-
chaperone, are part of the IKK complex and are responsible for its stabilization:
treatment with hsp90 inhibitor Geldanamycin (GA) prevents activation of NF-kappaB,
upon TNFα stimulation [35]. It is likely that the biological significance of IKK complex
regulation by hsp90 and other chaperones is to provide a flexible mechanism to co-
regulate a variety of stress response in collaboration with other signaling pathways,
including immune regulation during heat shock [36]. Considering that, we are planning
to figure out if morgana can interact with the IKK complex together with hsp90 and if
there are differences in the activity of the complex itself upon morgana
downregulation or overexpression, in order to define more clearly morgana role in this
pathway.
Furthermore, another major point that we are planning to do is to evaluate if
morgana expression correlates with NF-kappaB pathway hyperactivation in breast
cancer patients. For instance, we would like to analyze the expression of some NF-
kappaB target genes that we found overexpressed in cells with high morgana levels. In

27. 27
accordance, we show by immunohistochemical analysis that section of primary tumors
developed by mice injected with control cells displayed high levels of IL-6, which is a
well-known target of NF-kappaB, while tumors developed by mice injected with cells in
which morgana was downregulated displayed a decrease in the amount of IL-6.
Furthermore, morgana correlates with tumor grade, lymph node positivity, and
proliferation capacity, which are all characteristics of aggressive tumors [7] and
morgana coding gene was found to be amplified in 19% of basal-like triple negative
breast cancers present in the Cancer Genome Atlas [37]. In accordance, morgana is
overexpressed at higher levels in triple-negative breast cancers (36%) [7] and it is well
known that these cancers are associated with a constitutive activation of the NF-
kappaB pathway: for this reason, we think that morgana will probably correlate with
hyperactivation of this pathway in TNBCs and we propose morgana as a marker for
aggressiveness.
To conclude, we demonstrated that morgana is overexpressed in highly
aggressive tumors and can promote invasion by enhancing MMP-9 expression through
activation of the NF-kappaB transcription factor. Thus, we show that morgana is a key
player in the metastasization process and we propose it as a new biomarker to
guarantee patience survival and precise targeted therapy, especially among triple-
negative breast cancers.

28. 28
ACKNOWLEDGMENTS
I wish to express my sincere thanks to Professor Mara Brancaccio, for passing on her
scientific passion to me and for making me grow not only as a scientist, but also as a
person. Moreover, I would like to thank Professor Guido Tarone, for the support and
guidance and for all the scientific advices. Importantly, my deep appreciation to
Fiorella Altruda and Lorenzo Silengo, for giving me the opportunity to develop my
thesis project at the Molecular Biotechnology Center and for providing me all the
facilities being required.
This work would not have been possible without the continuous and constant help of
my tutor, Federica Fusella, who patiently assisted me in every experiment I performed
and for her sincere and valuable guidance and encouragement. Moreover, I am
grateful to all the members of my group, Stefania, Laura, and Enrico, because of the
great atmosphere that we created together in the lab and for sharing expertise among
each other.
Furthermore, I would like to thank Francesca Orso from Daniela Taverna’s group for
her help with the Luciferase Assay, and to Tiziana Cravero from Emilia Turco’s group,
for the production of our morgana antibody for immunohistochemistry.
Eventually, I take this opportunity to express gratitude to my family and friends, for
economical and emotional support through all these years.

29. 29
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33. 33
FIGURES AND TABLES
Figure 1
Figure 1. Morgana knockdown cells show impaired migration and invasion in vitro. (A)
Western blot showing morgana downregulation in MDA-MB-231. VINCULIN has been used as a
control. (B) MDA-MB-231 cells infected with an empty vector (EMPTY) or with a shRNA
targeting morgana (shMORG1) were subjected to wound healing assay: representative images
at time points 0 and 24h (on the left); percentage of wound closure after 24 hours, calculated
using the AxioVision program (on the right). (C) Transwell migration assay on EMPTY or
shMORG1 MDA-MB-231 cells. (D) Transwell matrigel invasion assay on MDA-MB-231 infected
with an empty vector (EMPTY) or with two shRNAs targeting morgana (shMORG1, shMORG2).

34. 34
Table 1. Morgana knockdown cells show impaired metastasis formation in vivo. Counts of
micro-metastasis found in organs of NSG mice injected with MDA-MB-231 infected with an
empty vector (EMPTY) or shRNA targeting morgana (ShMORG1).

35. 35
Figure 2
Figure 2. Morgana knockdown cells show impaired metastasis formation in vivo. (A) Tail vein
injection of NSG mice with MDA-MB-231 infected with an empty vector (EMPTY) or with a
shRNA targeting morgana (shMORG). Representative images of hematoxylin stained lung
sections of NSG mice, 1 month after tail vein injection. (B) Representative images of lung
macro and micrometastasis developed by NSG mice 1 month after tail vein injection, stained
for morgana. (C) Metastasis formation 5 weeks after subcutaneous injection of MDA-MB-231
infected with an empty vector (EMPTY) or with a shRNA targeting morgana (shMORG1).

36. 36
Figure 3
Figure 3: Morgana downregulation causes a decrease in MMP-9 activity and expression.
Extracellular (A) and intracellular (B) activity of MMP-9 in MDA-MB-231 infected with an empty
vector (EMPTY) or shRNAs targeting morgana (ShMORG1 and ShMORG2) was evaluated by
gelatin zymography. (A) Top panel: representative zymogram of extracellular MMP-9 activity
(92KDa). Bottom panel: quantification of MMP-9 activity from three independent experiments
(B) Top panel: representative zymogram of intracellular MMP-9 activity (92KDa). Bottom
panel: quantification of MMP-9 activity from three independent experiments. (C) Western blot
analysis of conditioned medium (C.M.) obtained from MDA-MB-231 EMPTY or ShMORG1
immunostained with MMP-9, MMP-2 and hsp90 as loading control. (D) Western blot analysis
of total protein extracts (T.E.) from MDA-MB-231 EMPTY and ShMORG1 immunostained for
MMP9, morgana and vinculin as loading control.

37. 37
Figure 4
Figure 4. Morgana downregulation causes a decrease in MMP-9 activity and expression. (A,
B) Immunofluorescence for MMP-9 (green) (A) and MMP-2 (green) (B) on control (EMPTY) and
morgana downregulated (shMORG) MDA-MB-231 cells. DAPI was used to stain nuclei. (C, D)
RNA was extracted from MDA-MB-231 infected with an empty vector or shRNAs targeting
morgana and real-time PCR analysis was performed to analyze mRNA levels of morgana and
MMP-9 gene (C) and of morgana and MMP-2 gene (D). Results were calculated as fold changes
(mean ± s.e.m.) relative to controls, normalized on 18S.

38. 38
Figure 5
Figure 5. Morgana effect on MMP-9 expression is not dependent on ROCK/PTEN/AKT
pathway. (A) Western blot analysis of VINCULIN, MORGANA, P-MLC2 and total MLC2 on MDA-
MB-231 protein extracts. Cells were infected with an empty vector (EMPTY) or with a shRNA
against morgana (shMORG), and treated with 2µM Y-27632, for 24 hours. (B) Western blot
analysis of VINCULIN, MORGANA, MMP9, P-AKT and total AKT on MDA-MB-231 protein
extracts. Control cells (EMPTY) and morgana downregulated cells (shMORG) were treated with
5µM VO-OHpic, for 30 minutes. (C) RNA was extracted from control and morgana
downregulated MDA-MB-231, untreated or treated with 2µM Y-27632 for 24 hours. Then,
qRT-PCR was performed to analyze MMP9 mRNA. (D) RNA was extracted from control and
morgana downregulated MDA-MB-231, untreated or treated with 5µM VO-OHpic for 24 hours.
Then, qRT-PCR was performed to analyze MMP9 mRNA. Results were calculated as fold-
changes (mean + s.e.m.) relative to controls, and normalized on 18S.

39. 39
Figure 6
Figure 6. Morgana regulates MMP-9 expression through NF-kappaB transcription factor.
(A)Luciferase assay of AP-1 activity on control (EMPTY) and morgana downregulated MDA-MB-
231 cells (shMORG1, shMORG2), transfected with an empty vector (pGL3) or with an AP-1
responsive construct (3x-AP1). (B) Luciferase assay of NF-kappaB activity on control and
morgana downregulated MDA-MB-231 cells, transfected with an empty vector (pGL2) or with
and NF-kappaB responsive construct (3x-kB). (C) Luciferase assay of NF-kappaB activity on
control and morgana downregulated BT-549 cells, transfected with an empty vector (pGL2) or
with and NF-kappaB responsive construct (3x-kB). (D) Luciferase assay of NF-kappaB on control
and morgana overexpressing MCF-7 cells, transfected with an empty vector (pGL2) or with and
NF-kappaB responsive construct (3x-kB). (E) Luciferase assay of NF-kappaB on control (EMPTY)
and morgana overexpressing (OVER MORGANA) MCF10A cells, transfected with an empty
vector (pGL2) or with NF-kappaB responsive construct (3x-kB). All data are presented as fold
changes (mean + s.e.m.) relative to control cells, and normalized to Renilla activity.

40. 40
Figure 7
Figure 7. Morgana overexpressing cells show increased expression of NF-kappaB target
genes. (A) qRT-PCR of MMP-9, CCL-5, IL-1A, IL-1B, IL-6, and IL-24 genes on MDA-MB-231 cells
infected with an empty vector (EMPTY) or with two different shRNA targeting morgana
(shMORG1, shMORG2). (B) qRT-PCR of MMP-9, IL-1A, IL-1B, CCL-5, TGFβ genes on control and
morgana overexpressing MCF-7 cells, untreated or treated (with 10nM TNFα for 4 hours. (C)
qRT-PCR of MMP-9, IL-1A, IL-1B, CCL-5, TGFβ genes on control and morgana overexpressing
MCF-7 cells, untreated or treated with 10nM TNFα for 4 hours.

41. 41
Figure 8
Figure 8. Morgana overexpressing cells show increased expression of NF-kappaB target
genes. (A) Immunohistochemical analysis on cell pellets of control (EMPTY) and morgana
downregulated MDA-MB-231 (shMORG), stained for morgana and IL-6. (B)
Immunohistochemical analysis on sections of primary tumors developed by mice tail vein
injected with control (EMPTY) and morgana downregulated (shMORG) MDA-MB-231, stained
for morgana and IL-6.

42. 42
Figure 9
Figure 9. Morgana regulates the NF-kappaB pathway. (A) Immunoblotting of VINCULIN, P-
IKBα, total IKBα and MORGANA in control (EMPTY) and morgana downregulated (shMORG)
MDA-MB-231 cells. (B) Immunoblotting of VINCULIN, IKKβ, P-IKBα, total IKBα and MORGANA
in control (EMPTY) and morgana downregulated (shMORG) BT-549 cells. (C) Immunoblotting of
VINCULIN, P-IKBα, total IKBα and MORGANA in control (EMPTY) and morgana overexpressing
(MORG) MCF-7 cells. (D) Immunoblotting of VINCULIN, αTUBULIN, P-IKBα, total IKBα and
MORGANA in control (EMPTY) and morgana overexpressing (MORG) MCF10A cells.