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Understanding and controlling mitotic errors leading to aneuploidy in early ovarian cancer development

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Understanding and controlling mitotic errors leading to aneuploidy in early ovarian cancer development

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Content UNDERSTANDING AND CONTROLLING MITOTIC ERRORS LEADING TO
ANEUPLOIDY IN EARLY OVARIAN CANCER DEVELOPMENT

Theresa M. Austria

A Dissertation Presented to the
FACULTY OF THE GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA

In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
GENETIC, MOLECULAR AND CELLULAR BIOLOGY

May 2019

Copyright 2019 Theresa M. Austria
ii

DEDICATION

This dissertation is dedicated to the educators in my life, especially Mrs. Sharon Harrison and
Mrs. Jennifer Tentes. Thank you for instilling a love of learning in me and encouraging me to
pursue a career in science. You have been such uplifting role models to me and because of you, I
wear the term “nerd” as a badge of honor and distinction.

iii

ACKNOWLEDGEMENTS

I would like to take this opportunity to thank my family, especially my mother, Teresita Austria,
for their love and support as I worked to achieve this degree. Mom, thank you for your patience,
encouragement, and understanding as I inched my way towards this milestone. It was a long road,
much longer than even I anticipated, but you stuck by me the whole way through. To my siblings
and extended family: thank you for keeping me grounded. There is nothing like the feeling of being
“home” and forgetting the stress of school, even for a moment. It has been my sanity through the
tough times.

To my late father, Lamberto Austria, and my late grandfather, Benigno Mendoza: thank you for
watching over me. I carry your words of reassurance and confidence with me, particularly on the
dark days. Dad, thank you for believing that I would be able to hold my own as an adult as I
ventured onto unknown ground. It instilled a sense of confidence and self-reliance in myself.
Tatay, thank you for always being my champion. You, above all, made me believe that I could do
anything.

To my research mentor, Dr. Louis Dubeau: thank you for recognizing my potential and sculpting
me into the scientist I have become. Even when I doubted myself, you seemed to see more in me
than I saw in myself. Thank you. To my committee members, Dr. Michael Stallcup, Dr. Ebrahim
Zandi, and Dr. Judd Rice: thank you for your guidance. You have pushed me to become a better
scientist than I thought I could be.

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To my lab mates, both past and present, Dr. Ying Liu, Dr. Elena Enbom, Dr. Christina Marion,
Emily Zhang, and William Wu: thank you for making this lab a second home. Thank you for your
input and support through the ups and downs of this difficult yet rewarding experience. Thank you
for being there for the early mornings, late nights, and long days (including weekends and
holidays). Thank you for indulging me in intellectual repartee, problem-solving and
troubleshooting, scientific development and planning, as well as stress venting. You have made
being part of this lab more than just bearable; you have made it enjoyable.

To the California State University, Long Beach President’s Scholars Program, McNair Scholars
Program, and RISE Program: thank you for all the opportunities you have given me to get me to
this point. I might not have made it without your support. A special thank you to Valerie Bordeaux,
Jennifer Hurley, Dr. Howard Wray, Denise Davis, the late Bob Rogers, Lily Salter, and Dr. Marco
Lopez. The skills and opportunities that you have afforded me are immeasurable and I will never
take them granted. To my undergraduate mentor Dr. Mason Zhang and lab manager Gayle Boxx,
thank you for giving me my first experience in research. Go Beach!

To all those who have touched my life: I hope I have made and to continue to make you proud.

Fight On!

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TABLE OF CONTENTS
DEDICATION ................................................................................................................................ ii
ACKNOWLEDGEMENTS ........................................................................................................... iii
TABLE OF FIGURES .................................................................................................................. vii
ABBREVIATION GLOSSARY ................................................................................................. viii
ABSTRACT .................................................................................................................................... 1
CHAPTER 1: INTRODUCTION ................................................................................................... 3
Ovarian cancer .................................................................................................................... 3
Familial ovarian cancer ....................................................................................................... 5
BRCA1 ................................................................................................................................ 6
BRCA1 and Ovarian Cancer ............................................................................................... 8
Cell non-autonomous mechanism ....................................................................................... 9
Aneuploidy and ovarian cancer ........................................................................................ 12
Cell model for aneuploidy development in ovarian cancer cells. ..................................... 12
The Cell Cycle .................................................................................................................. 13
Cell cycle regulation ............................................................................................. 14
G0: a non-proliferative cell state ........................................................................... 16
G1 and the G1/S Checkpoint, The Restriction Point ............................................ 17
S-phase: DNA Replication .................................................................................... 19
G2 and the DNA damage checkpoint ................................................................... 21
Mitosis and the Mitotic Checkpoint ...................................................................... 22
The G/M transition ................................................................................................ 23
Prophase ................................................................................................................ 24
Prometaphase, the Spindle Assembly Checkpoint, and the Mitotic Checkpoint
Complex ................................................................................................................ 24
Metaphase ............................................................................................................. 28
Anaphase ............................................................................................................... 29
Telophase and Cytokinesis ................................................................................... 30
Rationale ........................................................................................................................... 32
CHAPTER 2: MECHANISM OF CYTOKINESIS FAILURE IN OVARIAN
CYSTADENOMAS WITH DEFECTIVE BRCA1 AND P53 PATHWAYS ............................. 34
INTRODUCTION ............................................................................................................ 34
MATERIALS AND METHODS ...................................................................................... 36
RESULTS ......................................................................................................................... 40
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Mechanism of metaphase arrest in aging ovarian cystadenomas ......................... 40
Fate of arrested cells ............................................................................................. 42
Consequences of BRCA1 down-regulation .......................................................... 43
DISCUSSION ................................................................................................................... 47
CHAPTER 3: AURORA A KINASE REGULATES CHROMOSOME CONDENSATION
WITH CONSEQUENCES FOR METAPHASE TO ANAPHASE TRANSITION .................... 60
INTRODUCTION ............................................................................................................ 60
MATERIALS AND METHODS ...................................................................................... 62
RESULTS ......................................................................................................................... 65
Loss of Aurora A leads to an increase in cohesion fatigue. .................................. 65
Loss of Aurora A inhibits proper chromosome condensation .............................. 66
Aurora A regulates Histone H3 phosphorylation.................................................. 67
DISCUSSION ................................................................................................................... 67
CHAPTER 4: SUMMARY AND FUTURE DIRECTIONS........................................................ 73
REFERENCES ............................................................................................................................. 80

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TABLE OF FIGURES

Figure 1: Domain map of BRCA1. ............................................... Error! Bookmark not defined.
Figure 2: Reduced microtubule anchoring in aging ovarian cystadenomas ................................ 52
Figure 3: Mechanism of escape from the spindle assembly checkpoint in cells with reduced
BRCA1 expression ....................................................................................................... 54
Figure 4: Consequences of BRCA1 down-regulation on formation of bridges between
chromosome plates ....................................................................................................... 56
Figure 5: Consequences of BRCA1 down-regulation on cytokinesis failure .............................. 57
Figure 6: Working model for interplay between cell-nonautonomous and cell-autonomous
mechanisms of cancer predisposition in BRCA1 mutation carriers ............................. 58
Figure 7: Aurora A downregulation leads to cohesion fatigue .................................................... 71
Figure 8: Aurora A downregulation leads to defects in chromosome condensation ................... 72
Figure 9: Working model for prevention of aneuploidy development by interruption of the
interplay between cell-nonautonomous and cell-autonomous mechanisms of cancer
predisposition in BRCA1 mutation carriers by Aurora A inhibition ............................ 78

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ABBREVIATION GLOSSARY

ALIX – ALG-2-interacting protein, also known as programmed cell death 6-interacting protein
(PDCD6-interacting protein), involved in cytokinesis
APC/C – Anaphase promoting complex/cyclosome, a ubiquitin ligase that targets proteins for
degradation to promote mitotic progression from metaphase to anaphase
ATM – Ataxia-telangiectasia mutated, a serine/threonine kinase involved in DNA damage
repair
ATP – Adenosine triphosphate, coenzyme used as an energy carrier in the cells
ATR – Ataxia telangiectasia and Rad3 related, a protein involved in DNA damage repair
BARD1 – BRCA1-associated RING domain protein 1, interacts with BRCA1 to form a
heterodimer which helps relocate BRCA1 to the site of DNA damage for DNA damage
repair
BRCA1/2 – italicized: Breast cancer type 1/2 gene that is often mutated in hereditary breast and
ovarian cancer, nonitalicized: Breast cancer type 1/2 susceptibility protein, encoded by
the BRCA1/2 gene and is responsible for DNA damage repair of double strand breaks
BRCT – BRCA1 C terminus domain, predominantly found in proteins involved in DNA damage
response and repair
BRIP1 – BRCA1 interacting protein 1 gene, encodes Fanconi anemia group I protein involved
in DNA double strand repair
BUB1/3 – budding uninhibited by benzimidazoles 1/3, BUB1 is a mitotic checkpoint serine
threonine-protein kinase that regulates MCC formation; BUB3 is mitotic checkpoint
protein that is a component of the MCC which regulates the SAC
ix

BUBR1 – BUB1-related protein kinase, mitotic checkpoint serine threonine-protein kinase that is
a component of the MCC which regulates the SAC, also known as BUB1b or
BUB1beta
CA-125 – cancer antigen 125, carcinoma antigen 125, carbohydrate antigen 125, also known as
mucin 16, used as a tumor biomarker for ovarian cancer
CAK – CDK-activating kinase, phosphorylates Thr160 in the CDK activation loop to activate
the cyclin-CDK complex
CDC – Cell division cycle protein, also knowns as CDK (with exception of CDC20)
CDH1 – APC/C activator protein, an adaptor protein of the APC/C that confers substrate
specificity
CDK – cyclin dependent kinase, interacts with cyclins to regulate cell cycle progression
CHEK2 – checkpoint kinase 2, Italicized: gene that encodes CHK2 protein, mutations in this gene
are often associated with cancers
CHK2 – Checkpoint kinase 2, serine threonine kinase involved in DNA damage response that
is involved in DNA repair, cell cycle arrest, and apoptosis
CIP – CDK interacting protein, CIP and KIP together comprise a family of CKIs
CKI – cyclin dependent kinase inhibitor, inhibits cyclin-CDK complex activity to halt cell
cycle progression
CKS – cyclin dependent kinase subunit, confers/alters specificity of CDK/cyclin interaction as
CDKs can interact with multiple cyclins.
COBRA – Cofactor of BRCA1, also known as NELF-B
CPC – chromosomal passenger complex, is involved in the correction of microtubule
attachments
x

DAP – death-associated protein, a multi-functional protein involved in transcription, cell cycle
regulation, and apoptosis.
DAPI – 4’,6-diamidino-2-phenylindole, a fluorescent stain that binds to AT rich regions in the
DNA for use in fluorescence microscopy
Dlk – Death-associated protein (DAP)-like kinase, may be involved in Histone H3 Thr11
phosphorylation
DNA – deoxyribonucleic acid, the carrier of genetic information
DTT – dithiothreitol, a reducing agent used to break disulfide bonds
E2F – transcription factors that regulate transcription of proteins involved in DNA synthesis,
inhibited by hypophosphorylated RB protein
EB1 – End binding protein 1, + end microtubule binding protein that accumulates at
kinetochores upon microtubule anchoring.
EDTA – ethylenediaminetetraacetic acid, chelating agent that sequesters metal ions, specifically
calcium and iron
EOC – epithelial ovarian cancer, accounts for the majority of ovarian cancers
ESCRT – endosomal sorting complexes required for transport, remodels the membrane by
bending it which is important for abscission during cytokinesis and the formation of
vesicles
FBS – fetal bovine serum, nutrient component used in cell culture.
G1 – Gap 1, growth phase of the cell cycle after mitosis
G2 – Gap 2, growth phase of the cell cycle after DNA synthesis
Gadd45 – Growth arrest and DNA damage, involved in genotoxic and physiological stress
response
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GAPDH – glyceraldehyde 3-phosphate dehydrogenase, enzyme involved in glycolysis, used as a
protein loading control for western blot
GFP – Green fluorescent protein, this protein is not naturally found in mammalian cells, so
siRNA was designed against this sequence
GTP – guanosine-5‘-triphosphate, important for RNA synthesis during transcription
H2AX – Histone H2A, member X, involved in nucleosome formation
HEPES – 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, a zwitterionic organic chemical
buffering agent.
IgG (H+L) – Immunoglobulin G, Heavy and light chain, full length antibody used in western blot
INCENP – inner centromere protein, regulatory component of the CPC
INK4 – a family of CKIs that inhibit CDK4 blocking cell cycle progression during the
restriction point
kDa – Kilodalton, 1000 unified atomic mass units, unit of size measurement for proteins
KIP – Kinase inhibitory proteins, CIP and KIP comprise a family of CKIs
kMT – kinetochore microtubules, refers to microtubules that are bound to kinetochores as
opposed to microtubules that form the mitotic spindle
M – mitosis, phase of active cell division
MAD2 – mitotic arrest deficient 2, component of the MCC which regulated the SAC
MAPK – mitogen activated protein kinase, regulates functions involved in cell proliferation,
gene expression, and mitosis.
MCC – Mitotic checkpoint complex, regulates the SAC
MEM – minimum essential media, used for cell culture
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MG132 – a potent reversible and cell permeable proteasome inhibitor, used to arrest mitotic cells
at metaphase
MgcRacGAP – a rac GTPase activating protein. Plays an important role in cytokinesis by
regulating cortical movement through RhoA
MKLP1 – Mitotic kinesin-like protein 1, a component of centralspindlin that has been implicated
in the assembly of the midzone/midbody during mitosis and is essential for cytokinesis.
MPF – maturation promoting factor, composed of CDK1 and cyclin B
MUTYH – MutY homolog, encodes DNA glycosylase MUTYH glycosylase which is involved in
oxidative DNA damage repair
NBS1 – Nibrin, involved in double strand break repair
NDC80 – Kinetochore protein NDC80 homolog, component of the outer kinetochore
NEBD – Nuclear envelop breakdown, dissolution of the nuclear envelope that occurs during
mitosis
NELF – negative elongation factor, protein that negatively impacts transcription by pausing
RNA polymerase II 20-60 nucleotide downstream of the transcription start site.
NEM – N-ethylmaleimide, deSUMOylase inhibitor
OPG – osteoprotegrin, also known as osteoclastogenesis inhibitory factor (OCIF) or tumor
necrosis factor receptor superfamily member 11B (TNFRSF11B), tumor necrosis
factor (TNF) cytokine receptor that can regulate bone density by acting as a decoy
receptor for RANKL and inhibit apoptosis by inhibition of the TNF-related apoptosis
inducing ligand (TRAIL) induced apoptosis pathway
PALB2 – encodes partner and localizer of the BRCA2 gene, recruits BRCA2 and RAD51 to
DNA breaks in homologous recombination repair
xiii

PAO – Phenylarsine oxide, inhibits internalization of cell surface receptors, inhibits tyrosine
phosphatases, with no effect to tyrosine kinase, also a metabolic poison.
PARP – poly (ADP-ribose) polymerase, involved in single strand break repair
PBS – Phosphate buffered saline, a water-based salt solution commonly used in biological
research
PI3-kinase – phosphoinositide 3 kinase, phosphorylates the hydroxyl group at the 3 position of the
inositol ring of phosphatidylinositol
PIKK – phosphatidylinositol 3-kinase-related kinase, a family of Ser/Thr kinases with sequence
similarity to PI3K, includes ATM, ATR, PRKDC (Protein Kinase, DNA-Activated,
Catalytic Polypeptide), MTOR (Mammalian Target of Rapamycin), SMG1
(Suppressor of Morphogenesis in Genitalia), and TRAPP (Transformation
/Transcription Domain-Associated Protein)
PMSF – phenylmethane sulfonyl fluoride, serine protease inhibitor
PP1/2 – protein phosphatase 1/2
PRC1 – protein regulator of cytokinesis 1, involved with midzone microtubule formation
PTEN – phosphatase and tensin homolog, dephosphorylates the 3’ phosphate of
phosphatidylinositol (3, 4, 5)- trisphosphate (PIP3) forming of phosphatidylinositol (4,
5)- bisphosphate (PIP3)
PVDF – polyvinylidene fluoride, a highly non-reactive thermoplastic fluoropolymer composed
of polymerized vinylidene difluoride, membranes of this substance are used to
immobilize proteins for use in western blots as it has a non-specific affinity for amino
acids
RAD50 – encodes DNA repair protein involved in DNA double strand break repair
xiv

RAD51C – encodes RAD51 homolog c, involved in DNA double strand break repair
RAD51D – encodes RAD51 homolog d, involved in DNA double strand break repair
RANKL – receptor activator of nuclear factor kappa-B ligand (also known as tumor necrosis
factor ligand superfamily member 11 (TNFSF11), TNF-related activation-induced
cytokine (TRANCE), osteoprotegerin ligand (OPGL), and osteoclast differentiation
factor (ODF), controls bone regeneration and remodeling, receptor for osteoprotegerin
which controls cell proliferation by altering levels of proteins such as cyclin D1
RB – retinoblastoma protein, regulates cell cycle progression at the restriction point that
controls transition for G1 to S phases as, in its unphosphorylated state, it binds and
inhibits function of E2F proteins which is responsible for the transcription of proteins
necessary for DNA replication
RING – Really Interesting New Gene, a protein structural domain of zinc finger type containing
a C3HC4 amino acid motif that binds 2 ZINC cations
RNA – ribonucleic acid, a nucleotide macromolecule which, in humans, is involved in the
expression of genes by coding for a protein product.
S phase – phase of the cell cycle where DNA is synthesized
SAC – Spindle assembly checkpoint, a checkpoint during mitosis that inhibits mitotic
progression un the presence of kinetochores that are unattached to microtubules.
Ser – Serine, an amino acid
siRNA – small interfering RNA (also known as short interfering RNA or silencing RNA), used
to decrease levels of a specified protein by complementary interaction with its mRNA
SMC – structural maintenance of chromosomes, an ATPase that are involved in higher order
chromosome organization, such as chromosome condensation
xv

STAG – stromal antigen, a subunit of cohesion complexes,
SV40 – simian vacuolating virus 40, SV40 large T antigen perturbs RB and P53 proteins
causing constitutive activation of the cell cycle and prevents DNA repair mechanisms,
respectively
SWI/SNF – SWItch/Sucrose Non-Fermentable, a nucleosome remodeling complex
TBS – Tris buffered saline, buffer employing use of Tris(hydroxymethyl)aminomethane
Thr – threonine, an amino acid
TLB – Triton lysis buffer, used for whole cell lysis
Tyr – tyrosine, an amino acid
UV – Ultra violet, a form of electromagnetic radiation that can cause DNA damage
Vps23 – Also know and TSG101, tumor susceptibility gene 101, interacts with ESCRT-I during
cytokinesis
ZIP – Zipper interacting protein, may be involved in Histone H3 Thr11 phosphorylation

1

ABSTRACT

An in vitro cell model of ovarian cancer development was previously developed and characterized
in our laboratory. Previous students have used this model to show that the cells reach a mitotic
arrest at the spindle assembly checkpoint as they proliferate and age in culture. Here, I show that
this mitotic arrest is due to defects in microtubule anchoring associated with decreases in BUB1,
the master regulator of the mitotic checkpoint complex formation. The prolonged arrest leads to
cohesion fatigue that primarily resolves in cell death, though a small portion of cells undergo
mitotic slippage where cellular structures at the site of cleavage furrow ingression interfere with
abscission leading to cytokinesis failure and tetraploidy. Our laboratory previously showed that
downregulation of BRCA1 in aging cell populations leads to cells overcoming the mitotic arrest,
but only at the expense of cytokinesis failure, generating binucleated tetraploid cells. I report here
that decreases in BRCA1 to levels similar to those present in human BRCA1 mutation carriers lead
to an increase in microtubule anchoring, which may lead to erroneous attachment to the
kinetochore resulting in severe intra-chromosomal bridging. Severe bridging results in cytokinesis
failure contributing to tetraploidy and subsequently aneuploidy development, which may
significantly contribute to cancer predisposition in BRCA1 mutation carriers. I also investigated
Aurora A, a serine/threonine kinase that is upregulated in many cancers whose protein levels are
inversely related to that of BRCA1, for its role in mitotic regulation. Previous students in our
laboratory have shown that loss of Aurora A leads to mitotic arrest followed by cell death leading
to a decrease in proliferating tetraploid cell populations. Here, I show that loss of Aurora A leads
to an increase in cohesion fatigue and defects in chromosome condensation due to loss of
phosphorylation on sites of Histone H3 that are important for chromosome condensation. I
2

hypothesize that this defect in chromosome condensation leads to chromosome entanglement that
would prevent chromosome segregation leading to cohesion fatigue resulting in cell death.
3

CHAPTER 1: INTRODUCTION

Ovarian cancer
Ovarian cancer is the fifth most common cause of cancer-related deaths in women and is
the deadliest form of the gynecological cancers despite only being the 11
th
most common form of
cancer in women (1). According to the American Cancer Society, approximately 22,240 women
will be newly diagnosed with ovarian cancer and approximately 14,070 women will die from it in
2018. The risk of a woman developing ovarian cancer in her lifetime is 1 in 78 and her lifetime
chance of dying from ovarian cancer is 1 in 108. Ovarian cancer is primarily diagnosed in older
women, with the majority of women who are diagnosed with ovarian cancer being 63 years or
older. Fortunately, incidence of new ovarian cancer cases has steadily fallen approximately 1.5%
per year in the past 20 years and deaths have dropped by 2.3% on average between the years of
2006 and 2015. While 5-year survival for ovarian cancer is relatively good if found to be localized
or regional, 92.3% and 74.5%, respectively, ovarian cancer overall has a poor prognosis as nearly
60% of cases are discovered when the cancer has metastasized (2).
Reasons why ovarian cancers are often not discovered until they reach the late stages of
the disease are that the symptoms of the disease are vague and can be mistaken for menstrual
symptoms and because there currently are no reliable screening methods for early detection (3-5).
Early stage ovarian cancer is often asymptomatic resulting in detection in the late stages. Signs
and symptoms of ovarian cancer may include abdominal swelling or bloating, quickly feeling full
while eating, weight loss, pelvis discomfort, changes in bowel movements including constipation,
and frequent need to urinate. Use of CA-125 blood tests and transvaginal ultrasounds to detect
ovarian cancer found more cancers but the outcomes were overall no different than women who
4

have not been screened: those who were screened using these techniques did not live longer than
those who were not screened nor were they less likely to die from ovarian cancer (6, 7)
Additionally, CA-125 testing often resulted in false positives (1). Factors that can contribute to
a woman’s increased risk for ovarian cancer include age, obesity, hormone therapy, and number
of menstrual cycles due to early menarche and late menopause, and family history of ovarian,
breast, and colorectal cancer while full term pregnancy prior to the age of 26, multiple parity, fewer
menstrual events, breast feeding, use of oral birth control, tubal ligation, and removal of one’s
ovaries and/or uterus decreased one’s risk for developing the disease (1, 8-11). However, there are
negative implications for these risk-reducing procedures (12, 13). Woman at increased risk for
ovarian cancer development may choose to not receive or postpone risk-reduction surgery as the
consequence is infertility. Additionally, loss of the ovaries leads to a decrease in estrogen
production, which can lead to osteoporosis, cardiovascular disease, and menopause symptoms.
There are 4 major histological subtypes of ovarian carcinomas: serous, mucinous,
endometroid, and clear cell which account for 85-90% of ovarian malignancies (1, 14). The serous
subtype is further subdivided into high grade and low grade. There is evidence that, although
ovarian carcinoma development is driven by the ovary, these tumors originate in derivatives of the
Müllerian ducts, including the fallopian tubes and other components of extra-uterine Müllerian
epithelium (15). There is a strong correlation between the high-grade serous subtype and BRCA1
gene abnormalities. Approximately 50% of ovarian carcinomas are serous carcinomas.
Endometrioid carcinoma is the second most common histotype of ovarian cancer, accounting for
approximately 25% of ovarian carcinomas and is also believed to arise from endometriosis.
Mucinous carcinoma account for approximately 10% of cases and the clear cell histotype accounts
for approximately 5% are the least common histotypes of ovarian cancer (14).
5

Several chemotherapies have been used to treat ovarian cancer (16, 17). Platinum-based
anti-neoplastic drugs carboplatin and cisplatin are DNA intercalators that are commonly used as
chemotherapy against ovarian cancer. These drugs work by inhibiting DNA replication. However,
there are some serious negative side-effects due to the use of these drugs including nephrotoxicity
and neurotoxicity, nausea and vomiting, ototoxicity, electrolyte disturbances, hemolytic anemia
(16). Regardless of type of therapy, most patients relapse and develop resistance (18). To increase
efficacy and decrease side-effects, targeted therapies have been developed. These include drugs
such as bevacizumab, pembrolizumab, and poly (ADP-ribose) polymerase (PARP) inhibitors.
Bevacizumab is a recombinant humanized monoclonal antibody that inhibits angiogenesis (19).
Pembrolizumab is a humanized antibody that targets programmed cell death one (PD-1) receptor
on lymphocytes inhibiting a protective mechanism used by cancer cells to evade immune
surveillance (20). PARP is involved in the repair of DNA single strand breaks. PARP inhibitors
prevent the repairs of these breaks and if these nicks cannot be repaired, DNA replication turns
these single strand breaks into double strand breaks, which, in cancers with BRCA1 mutations,
cannot be repaired leading to cell death (21-23).

Familial ovarian cancer
While most ovarian cancers are sporadic, familial predisposition accounts for 5-10% of
cases (1). Ethnic background carries a certain association with increased incidence of ovarian
cancer. Caucasians and peoples of Alaskan and Native American descent have the highest
incidence of ovarian cancer while non-Hispanic blacks and Asians and Pacific Islanders have the
lowest risk (2). Several cancer syndromes, such as Lynch syndrome, are associated with an
increased risk of the endometrioid subtype of ovarian carcinoma due to mutations in mismatched
6

repair genes, the PTEN tumor hamartoma syndrome, Peutz-Jeghers syndrome, and MUTYH-
associated polyposis (24, 25). Several other mutations associated with increased risk of ovarian
cancer including those in BRIP1, RAD51C, RAD51D, CDH1, CHEK2, PALB2 and RAD50 (26,
27).
By far the most common and most penetrant cause of familial ovarian carcinoma
predisposition is germline mutations in the BRCA1 or BRCA2 genes which increases a woman’s
lifetime risk of developing ovarian cancer by 15-45% for BRCA1 and 10-40% for BRCA2 (24, 25).
Mutations in the BRCA1 and BRCA2 genes are closely associated with high-grade serous epithelial
ovarian carcinomas, the most common subtype of ovarian cancer (1). Though mutations in these
genes both increase risk for breast and ovarian cancer, they are unrelated genes, i.e. they are not
homologs or paralogs.

BRCA1
BRCA1 is often regarded as a classical tumor suppressor gene, although work from our
laboratory and that of others have challenged this view. It is best known for its role in DNA repair,
but also carries out multiple cellular functions such as cell cycle control, DNA damage repair,
transcriptional regulation, maintenance of genome integrity, cell replication, recombination, and
chromatin hierarchical control (28, 29). BRCA1 is involved in DNA damage repair through the
homologous recombination repair pathway (30). In the presence of a double strand DNA break,
BRCA1 gets phosphorylated by ATM/ATR in the homologous recombination repair pathway
which induces a cell cycle arrest. BRCA1 also plays a role in mismatched and single strand repair
(30). BRCA1 also acts as a ubiquitin ligase during mitosis when bound to BARD1 where it
regulates microtubule nucleation and elongation (31).
7

The BRCA1 gene is located on the long arm of chromosome 17 at region 2 band 1 and
contains 24 exons (32). The BRCA1 protein has four major domains: a RING domain, a BRCA1
serine domain, and two BRCA1 C terminus (BRCT) domains (33) (Fig. 1). The proteins interacting
with the RING finger domain are characterized as E3 ligase enzymes that are involved in
ubiquitination, which includes BARD1. The serine cluster domain is the site of phosphorylation
on BRCA1 and is also subject to high rates of mutation. This site is also phosphorylated by
ATM/ATR kinases, which are involved in DNA damage repair. The serine residues may also play
a role in the localization of BRCA1 to DNA damage sites in the DNA damage response pathway.
The BRCT domain recognizes phosphorylated binding partners of BRCA1 that are mainly
involved in DNA damage response.

8

Figure 1: Domain map of BRCA1 (33). RING, serine containing domain (SCD), and BRCT
domains are indicated. Nuclear export signal (NES) and nuclear localizing sequence (NLS)
sequences are also depicted. Horizontal solid black lines indicate protein binding domains for the
listed binding partners. Red circles mark phosphorylation sites.

BRCA1 and Ovarian Cancer
Hundreds of mutations in the BRCA1 gene have been associated with increased risk of
breast and ovarian cancer. Mutations in the BRCA1 gene yield a non-functional protein, thus
inhibiting its role in DNA repair. Any DNA damage incurred in the absence of functional BRCA1
is inherited and accumulated in daughter cells promoting tumor growth. Mutation carriers of
certain genetic backgrounds tend to have a very specific inherited mutation due to a founder effect,
a mutation that has been passed down through generations. For instance, Ashkenazi Jewish patients
with inherited breast and/or ovarian cancer tend to have BRCA1 mutations 185del AG, 188dell11,
and 532insC (34, 35).
Epigenetics may also contribute to the role of BRCA1 in breast and ovarian cancer
development. Sporadic epithelial ovarian carcinomas (EOCs), which make up about 90% of
ovarian carcinomas, show significantly decreased expression of BRCA1 due to loss of
9

heterozygosity, promoter hypermethylation, or haploinsufficiency (36-39). However, the exact
significance of these epigenetic changes is not clear (40). Evidence of precancerous lesions in the
fallopian tubes of BRCA1 mutation carriers have also been found (41-43). BRCA1 expression has
been found to be repressed by microRNA, specifically miR-182. miR-182 has been found to be
overexpressed in 70% of serous intraepithelial and high grade serous ovarian carcinomas (44).
microRNA miR-9 has also been found to be increased in stage IV serious ovarian cancers and is
inversely related to BRCA1 levels (45).

Cell non-autonomous mechanism
Though BRCA1 mutation carriers carry this mutation in all cells, BRCA1 mutations have
only been linked to breast and ovarian cancers. This is likely due to a cell non-autonomous effect
of BRCA1 mutated cells on target organs, an idea that challenges the view that BRCA1 acts as a
classical tumor suppressor.
One of the major functions of the ovary is to produce hormones that affect cells other than
the cells that produce those hormones. These cells can be adjacent to these hormone-producing
cells or cells in distant organs. There are several cases in which BRCA1 mutant hormone-producing
cells in the ovary have differential effects on outside cells and organs compared to their wildtype
counterparts which may contribute to their role in disease development.
Understanding how BRCA1 mutations in these hormone-producing cells effects the
menstrual cycle is essential to understanding its role in ovarian cancer development as it is known
that the menstrual cycle is the greatest risk factor contributing to ovarian carcinogenesis.
Previously, our lab has shown that tissue specific knockout of Brca1 in mouse ovarian granulosa
10

cells, which plays a critical role in regulating the menstrual cycle through the hypothalamic-
pituitary-gonadal axis, causes prolongation of the proestrus phase of the estrus cycle, the murine
equivalent of the human menstrual cycle (46). Proestrus is characterized as the period of
endometrial and ovarian follicular development and cell proliferation. This extended duration of
the proliferative phase of the estrus cycle was linked to tumor predisposition in mice due to an
extended increased exposure to estrogen unopposed to progesterone, recapitulating the conditions
associated with sporadic ovarian cancer predisposition in humans. We have also found that mice
carrying a Brca1 knockout in granulosa cells developed cystic tumors in the ovaries and in the
uterine horns demonstrating that Brca1 mutations may indirectly influence tumor development
possibly through an effector secreted by granulosa cells (47). This mouse model was also used to
show that the increased circulating estradiol in mice harboring a Brca1 mutation in granulosa cells
leads to an increase in femoral trabecular thickness and femoral length which are known
consequences of chronic estrogen stimulation (48). Interestingly, it was also found that granulosa
cells in this mouse model have an increase in expression of olfactory receptor mRNA (49). In
mice, the estrus cycle is regulated by exposure to male scent. It was found that, after being rendered
anovulatory by unisexual isolation, mice harboring a Brca1 mutation resumed ovulatory activity
more readily than their wildtype counterparts when exposed to male scent from bedding potentially
increasing their readiness to complete an estrus cycle. Together, these data show us how BRCA1
mutations can influence the menstrual cycle and thus influence ovarian cancer development.
Importantly, these findings in mouse studies have also been supported by studies in
humans. In a collaborative study of women involved in the UK Familial Ovarian Cancer Screening
Study (UK FOCSS) who carry either BRCA1 or BRCA2 mutations or no mutation, the endometrial
thickness was assessed using scans taken during each day of the menstrual cycle as a measurement
11

of hormone regulation and correlated these measurements to serum levels of estradiol and
progesterone (50). This study found that endometrial thickness in women carrying BRCA1/2
mutations was thicker than their nonmutant counterparts in the follicular phase and thinner during
the luteal phase. Additionally, median levels of progesterone and estradiol during the luteal phase
was higher than nonmutant carriers. Overall, this study found that BRCA1/2 mutation carriers are
exposed to higher levels of estradiol and progesterone which regulates endometrial thickness
during the menstrual cycle and these higher levels of hormone exposure are conducive to ovarian
carcinogenesis. In another collaborative study, serum levels of receptor activator of nuclear factor
kappa-B ligand (RANKL), a member of the tumor necrosis factor super family which plays a role
in bone remodeling and immune function, and its antagonist, osteoprotegerin (OPG), were
measured along with estradiol and progesterone in women in the UK FOCSS study during their
menstrual cycle and found that women carrying BRCA1/2 mutations, particularly mutations known
to be associated with increased breast cancer risk, had lower levels of OPG and progesterone (51).
Levels of OPG were inversely related to levels of mammary epithelial proliferation as determined
by Ki67 expression and this effect increased post menopause. This increase in proliferation due to
BRCA1 mutation status may contribute to cancer development in the breast.
Overall these data demonstrate the effect of BRCA1 mutation status on cell non-
autonomous regulation of target tissues including those contributing to breast and ovarian cancer.
Because hormonal regulation is a significant factor contributing to ovarian cancer, understanding
how the cell non-autonomous effects of BRCA1 mutations stimulate cellular proliferation is
essential to understanding ovarian cancer development.

12

Aneuploidy and ovarian cancer
While the signal that stimulates proliferation may play a role stimulating cancer
development, ovarian cancer is known as a disease of chromosomes. More than 95% of high-grade
serous ovarian carcinomas display aneuploidy, the presence of a chromosome number that is not
a multiple of the euploid number, the only major commonality in these cancers other than P53
mutations. Aneuploidy is important to ovarian cancer as it relates to poor prognosis, biological
aggressiveness, and tumor malignancy (52-54). Aneuploidy is believed to be an early event in
ovarian tumorigenesis (55). It has been well-established in the literature that aneuploidy
development can occur through a tetraploid intermediate (56-58). One way a tetraploid
intermediate can be generated is if a cell fails to complete mitosis after DNA replication. This can
occur by mitotic slippage or other mitotic errors. This mode of aneuploidy development is
important because ovarian cancer is near tetraploid, indicating that this mechanism of mitotic error
may play a role in be how aneuploidy develops in ovarian carcinogenesis. Thus, proliferation
stimulated by BRCA1 mutation status may increase chances for mitotic defects that can lead to
aneuploidy development.

Cell model for aneuploidy development in ovarian cancer cells.
Our lab has previously developed a longitudinal model of ovarian cancer development (59,
60). This model was created using ovarian cystadenomas that were transfected with SV40 Large
T antigen which binds and inhibits the proteins retinoblastoma (RB) and P53. Inhibition of P53
mimics the genetic background in which high-grade serous ovarian cancer develops. Inhibition of
RB causes constitutive activation of the cell cycle allowing cells to overcome senescence, or exit
13

from the cell cycle, and extends the cell population’s in vitro lifespan as RB controls cell cycle
progression.
As the cells age and continue to proliferate in culture, the cells inevitably undergo a mitotic
arrest. This arrest was determined to occur during the spindle assembly checkpoint which occurs
during prometaphase. The majority of these cells that are unable to overcome the mitotic arrest
and resolve in cell death. However, a portion of these cells may be able to overcome this mitotic
arrest by mitotic slippage. Cells that undergo mitotic slippage are unable to complete mitosis,
resulting in a cell with twice the amount of DNA and are often binucleated. This development of
tetraploidy and binucleation can create an environment of genomic instability that can promote
aneuploidy development.
In order to attain sustained cancer-like proliferation, these cells must overcome the last
mortality checkpoint which is cell crisis. Cell crisis is characterized as cell death due to telomere
attrition. After consecutive rounds of cellular reproduction, the ends of the DNA strands known as
telomeres shorten after every round of DNA replication. When these ends become too short, the
cell is unable to properly replicate the DNA, and this triggers cell death. Crisis can be overcome
by spontaneous activation of telomerase which would extend the length of the telomeres. Cells
that are able to overcome all these obstacles will have achieved replicative immortality, a key
characteristic of a carcinoma.

The Cell Cycle
As previously mentioned, aneuploidy development is critical for ovarian cancer
carcinogenesis. Aneuploidy development can occur through defects in cell cycle regulation. To
understand mechanisms by which aneuploidy can develop, we must understand how cellular
14

replication is regulated. Cellular division is a highly regulated and intricately orchestrated process
by which genetic content is duplicated and divided into 2 daughter cells. The execution by which
this information is passed on to the cellular progeny is crucial for genetic fidelity, ensuring that
the information is preserved and passed down without error. The consequences of errors in
duplication and/or inheritance of this genetic information can range from innocuous to fatal. To
ensure that cell replication occurs accurately and appropriately, cell division occurs under certain
conditions and has several checkpoints to safeguard against the propagation of genetic errors. The
cell cycle has been broken down into several phases with distinct events and functions

Cell cycle regulation
Progression through the cell cycle is controlled by phosphorylation steps catalyzed by
cyclin dependent kinase complexes (61-65). These complexes are comprised of a catalytic subunit,
called a cyclin dependent kinase (CDK), and its binding partner, a regulatory subunit known as a
cyclin. An additional regulatory subunit known as a cyclin-dependent kinase regulatory subunit
(CKS) binds the catalytic subunit of the CDK and helps the complex recognize its substrate. When
CDK complexes are active, cell cycle progression is promoted. When the CDK complexes are
inactive the cells cycle becomes arrested. Levels of CDKs are relatively constant throughout the
cell cycle, while levels of cyclins fluctuate depending on the phase of the cell cycle they are
needed. Additionally, the CDK’s function will depend on which cyclin it is bound to, as CDKs
have multiple cyclin binding partners. The activity of these CDK complexes is regulated by the
presence of cyclins, the presence of CDK inhibitors (CDKI), an activating phosphorylation, and 2
inactivating phosphorylations. Inhibitory phosphorylation on CDKs occur at Ser14 and Tyr15 by
wee-1 kinase and are eventually cleaved off by phosphatase CDC25 and an activating
15

phosphorylation on Thr161 is mediated by CDK-activating kinase (CAK) (62, 65). These protein
modifications help coordinate the timing of cell cycle progression by allowing cyclins to
accumulate before becoming active.
The cell cycle is broken down into 4 phases: G1 (Gap 1), S (DNA synthesis), G2 (Gap 2)
and M (mitosis) (66). Collectively the G1, S, and G2 phases of the cell cycle are considered the
interphase of the cell cycle while M phase is where actual cellular reproduction takes place.
Essentially, interphase prepares the cell for mitosis. The transition between these phases are
regulated by 2 checkpoints (timepoints in the cell cycle where the cell checks and confirms that
the cell and the environment are able to support the next step in the cell cycle): the G1/S checkpoint
(also known as the restriction point) and the G2/M checkpoint (DNA damage checkpoint) which
are controlled by cyclin dependent kinases (61-65). If a cell detects problems with the DNA or
other factors during these checkpoints, the cell will arrest itself until the problem is corrected or
even enter a non-proliferative phase known as G0. If the problem is unable to be corrected, the cell
will self-destruct to prevent incidence or propagation of genetic errors that could lead to disease.
The interaction of specific CDKs with their cyclins regulate progression through the four phases
of the cell cycle. In G1, the retinoblastoma (RB) protein is a critical target of phosphorylation by
CDKs. CDK targets in S phase are important for DNA synthesis. In G2/M, proteins involved
mitotic structures, such as the nuclear envelope and spindle assembly apparatus, are targets of
CDKs.
Overall, this tight regulation of cell cycle progression ensures that cells proliferate when
necessary and without error. Errors or defects in this regulation can have disastrous consequences
for the cells and can contribute to disease, such as cancer.

16

G0: a non-proliferative cell state
G0 is a phase where cells exit the active cell cycle of cell division to a non-cycling, non-
proliferative state. There are 3 states of G0: quiescent, differentiated, and senescent (67). Cell
quiescence is a reversible cell state of inactivity. These cells are characterized by low levels of
RNA production, and thus are not metabolically active. Cells can be stimulated to reenter the cell
cycle upon receipt of some external stimuli. In contrast, differentiated and senescent cell states are
irreversible forms of G0. Differentiated cells are the result of cellular programming by which stem
cells are programmed to differentiate into a terminal but functional non-proliferative state. These
cells can perform their cellular and metabolic functions indefinitely without reproducing. Should
these cells die, they are replaced by new terminally differentiated cells descended from stem cells.
Lastly, senescent cells are cells that do not reproduce due to having already undergone several
rounds of reproduction (68-70). Upon DNA replication, the ends of the DNA, known as telomeres,
shorten as the tip of the DNA strand is left out of replication. Telomeres, which are essentially
noncoding nucleotide repeats, protect chromosomes from erosion during replication and non-
specific fusing with other chromosome ends. Subsequent rounds of replication result in continued
telomere shortening. Eventually, extensive rounds of cellular replication cause the telomeres to
become too short and the cells stops dividing in order to prevent further telomere shortening. The
number of times that a cell can divide until the telomers become too short to withstand another
round of replication is known as the Hayflick limit (69). To prevent errors in DNA replication
when telomeres are significantly shortened, cells exit the cell cycle never to reproduce again but
may continue to carry out cellular processes.
Cell senescence becomes very relevant for diseases like cancer as unrestricted cellular
proliferation becomes subject to telomere attrition (68, 70). Continued proliferation despite
17

deteriorated telomers can lead to problematic consequences due to changes and damage to the
DNA. To circumvent this problem, most cancers can activate the genes that encode the telomerase
enzyme allowing cells to divide indefinitely. Telomerase is a reverse transcriptase that can
elongate the telomeres by adding nucleotides to the ends of the DNA strand. Telomerase is not
normally active or is active in low levels in most somatic cells. The telomerase enzyme uses an
RNA template to add nucleotides to the telomeres in a specific set of repeats. By adding to the
telomeres, cancer cells never reach their Hayflick limit. Cell that are able to escape senescence
may enter the first stage of the active cell cycle, G1.

G1 and the G1/S Checkpoint, The Restriction Point
G1 is the first phase of active cell cycling/replication. Called G1 or Gap 1 phase, this is a
stage of growth for the cell in preparation for the next stage of the cell cycle. Particularly, mRNA
and proteins are synthesized in preparation of cell division, such as histones that are used in DNA
synthesis. Cell growth is often limited to or a function of external or internal factors such as
external signals stimulating cell growth or division, availability of nutrients necessary to sustain
growth, and space of new cellular growth. Availability of nucleotide and amino acids necessary
for mRNA and protein synthesis is also a limiting factor of cell growth.
The transition from G1 to S phase, where the genome is replicated, is regulated by G1/S
checkpoint, known as the restriction point (also known as “The Point of No Return,”), and is the
first checkpoint in the cell cycle (71-73). It is believed that the restriction point is defective in
many, if not all, types of cancers (74). This checkpoint determines if the cellular environment is
supportive or favorable for cell cycle progression. Progression past the restriction point is
stimulated by the presence of mitogens, a substance that stimulates mitosis (72, 75, 76). Once this
18

restriction point is passed, the cell must complete the cell cycle; it cannot revert to early G1 or exit
the cell cycle and enter G0.
The restriction point is regulated by a tumor suppressor protein known as retinoblastoma
(RB) which is the target of CDK activity (72, 75, 76), When the RB protein is unphosphorylated
or hypo-phosphorylated, it binds transcriptions factors in the E2F family, such as E2F1, E2F2, and
E2F3, which are necessary for transcription of genes involved in DNA synthesis (74, 77, 78).
When RB is bound to an E2F protein, transcription of DNA synthesis genes is inhibited. If
conditions favor cell cycle progression, RB gets hyperphosphorylated by CDK complexes which
causes it to release E2F, freeing it for transcription, and thus the restriction point is passed, and the
cell cycle must be completed.
The G1 cyclins D and E and their associated CDKs regulate this checkpoint. D-type cyclins
D1, D2, or D4 interact with CDK4 or CDK6 and are responsible for initiating RB phosphorylation
while cyclin E-CDK2 complexes are responsible for hyperphosphorylation rendering RB unable
to continue to block cell cycle progression (79). Additionally, cyclin E causes assembly of the pre-
replication complex at the origin of replication needed during DNA replication in the following
phase. Stimulation by mitogens induces expression of cyclin D1 through the mitogen activated
protein kinase (MAPK) pathway and these mitogens can be endogenous or exogenous (cell
autonomous or non-autonomous) factors. Continued mitogenic activity leads to a decrease in CDK
inhibitors, reducing its activity allowing for progression past the restriction point. The mechanism
responsible for cyclin E-CDK complex activation has yet to be elucidated, though it is subsequent
to cyclin D-CDK complex activation. Mitogen stimulation must occur for at least two thirds of G1
in order to commit to the cell cycle and overcome this checkpoint.
19

CDK inhibitors help regulate the timing of cell cycle progression. CDK inhibitors in the
P16/INK4 family (P16
INK4
, P15
INKb4
, P18
INK4c
, and P19
INK4d
) inhibit CDKs by binding the CDK
catalytic domain preventing it from binding to cyclins, specifically that of CDK4 and 6. P21 or
CIP/KIP family inhibitors (P21
WAF1/CIP1
, P27
KIP1
, P57
KIP2
) work by binding the cyclin to the CDK
to inhibit their enzymatic activity, specifically G1 phase complexes cyclin D-CDK4/6 and cyclin
E-CDK2. High levels of p21 and p27 CDK inhibitors keep cyclin-CDK complexes inactive in cells
trapped in G0 (80).

S-phase: DNA Replication
The next phase of the cell cycle is the S phase where DNA is replicated or synthesized.
High fidelity replication of the DNA is essential for preventing genetic defects or abnormalities
that are detrimental to the cell’s normal function. In addition to the DNA, centrosomes are also
duplicated. The centrosomes are what will coordinate microtubule nucleation for mitosis.
During S phase, cyclin E is degraded, and cyclin A and cyclin B begin to accumulate (81,
82). As cyclin E is degraded, cyclin A binds to CDK2 replacing cyclin E. In this phase, cyclin A-
CDK2 ensures that the DNA is only replicated once by terminating the assembly of the pre-
replication complexes generated by cyclin E-CDK2 and preventing the assembly of additional
replication complexes by phosphorylating components of the DNA replication machinery (80, 83,
84). Cyclin A-CDK2 complex activity is regulated by P21 family CDK inhibitors. In late S-phase,
cyclin A associates with CDK1 (also known as cell division cycle protein 2, CDC2) and remains
associated with CDK1 from late S phase into late G2 phase where it is later replaced by cyclin B.
Interestingly, cyclin A-CDK1 is thought to activate and stabilize the cyclin B-CDK1 which in turn
promotes degradation of cyclin A through the ubiquitin ligase pathway (82).
20

The cell prevents more than one replication of the DNA by loading pre-replication
complexes at the replication origins during G1 which dissociates in S-phase once replication
begins (84-86). During DNA synthesis, DNA helicase unwinds the DNA double helix while DNA
polymerase base pairs free-floating DNA nucleotides to the separated DNA strands. DNA
polymerases are extremely accurate, with an intrinsic error rate of less than 1 mismatch for every
10
7
nucleotides (87). Additionally, some DNA polymerases are able to proofread the base-paired
sequences and remove mismatched nucleotides. If DNA damage is detected during DNA
synthesis, replication is halted through the ATM/ATR pathway until the damage can be corrected
(88).
During DNA replication the cell must ensure accurate duplication of the genome. Certain
intrinsic and extrinsic factors, such as the presence of transcription complexes and DNA damage
repair machinery, DNA damage due to ultraviolet (UV) radiation, and depletion of
deoxyribonucleotide triphosphates by hydroxyurea inhibition of ribonucleotide reductase can slow
DNA replication (89, 90). To cope with these issues, the cell can detect stalled replication forks
and respond accordingly by delaying mitosis until DNA replication is complete. There are 3 main
classes of checkpoint proteins: sensors which detect blocks in replication or DNA damage,
transducers that relay this signal, and effectors that act on the targets of the checkpoint (90). If
problems with DNA replication are detected, the cell cycle progression is halted, and effector
proteins set out to correct the problem.

21

G2 and the DNA damage checkpoint
The G2 phase follows DNA synthesis and is a period of rapid cell growth and protein
synthesis intended to prepare the cell for mitosis. This phase is important as transcription cannot
occur during mitosis, so all proteins needed during mitosis must be produced at this time.
Once the restriction point is passed and the DNA is replicated, the next checkpoint that
needs to be passed is the DNA damage checkpoint which ensures that the DNA is free damage,
ensuring the integrity of the DNA sequence before the cell is replicated. This checkpoint occurs at
the end of G2, however, DNA damage can be repaired during any point in interphase. This
checkpoint is controlled by the P53 protein, a transcription factor also known as “The Guardian of
the Genome” (91). In the presence of DNA damage, P53 causes the transcription of CDK inhibitors
and DNA repair genes which halts the cells cycle and corrects the damage to the DNA. This
checkpoint surveys for DNA damage due to UV radiation, oxidative stress, DNA intercalating
agents, and other causes of genotoxic stress. If DNA damage is detected, the transcription factor
P53 is activated. P53 then triggers the transcription of DNA damage repair proteins and CDK1 is
inhibited by p21, Gadd45, and 14-3-3σ (92). P21 sequesters inactive cyclin B1-CDK1 complexes
to the nucleus, 14-3-3σ sequesters active cyclin B1-CDK1 to the cytoplasm, and Gadd45 interferes
with cyclin B1-CDK interaction thus inhibiting cell cycle progression. Additionally, P53 represses
the transcription of CDK1. If the cell is unable to repair the DNA damage, P53 triggers the cell to
self-destruct by apoptosis so that the DNA damage does not get passed down to the cellular
offspring. Additionally, P53 can cause cell cycle arrest during both the G1 and G2/M phases of
the cell cycle.
Cell cycle arrest can also be achieved in a P53 independent manner through the ATM
(ataxia telangiectasia)/ATR (ATM and RAD3-related) pathway (93). ATM and ATR are members
22

of the phosphatidyl-inositol 3 kinase (PI3-kinase) like family of protein kinases (PIKKs). This
pathway is often involved in the correction of DNA damage due to UV light as well as stalled
replication forks and causes the phosphorylation of P53, CHK2 (checkpoint kinase 2), NBS1,
H2AX and BRCA1. This pathway causes the kinase CHK2 to phosphorylate and thus inhibit
CDC25 phosphatases which are responsible for dephosphorylating the inhibitory phosphate group
on CDKs. NBS1 (Nibrin) and H2AX (histone H2A, member X) are involved in the repair of double
strand breaks (94, 95).
Midway through G2, cyclin A begins to degrade and the cyclin B1-CDK1 complex, also
known as the maturation promoting factor (MPF), is accumulated (96, 97). When the level of this
complex reaches a certain threshold, the cell is ready to transition from G2 to mitosis. Inhibitory
phosphorylation on CDK1 at Ser14 and Tyr15 by wee-1 kinase allows accumulation of cyclin B
which is then cleaved off by phosphatase CDC25 allowing a rapid response. Additionally, an
activating phosphorylation on Thr161 is mediated by CDK-activating kinase (CAK) (98).
If the cell is able to overcome this checkpoint, the cell is ready for mitosis, the active phase
of cellular division.

Mitosis and the Mitotic Checkpoint
Mitotic regulation is extremely complex as no transcription takes place during mitosis, so
all proteins involved in mitotic regulation or their respective mRNA must be present at the
beginning of mitosis. Essentially, the function of the proteins involved in mitotic regulation are
controlled by localization, post-translational modifications such as phosphorylation, and protein-
protein interactions and mitotic progression is additionally regulated by the presence or
degradation of proteins. Mitosis is broken down into 4 (or 5) phases: prophase,
23

prometaphase/metaphase, anaphase, and telophase. For most cells, mitosis concludes with
cytokinesis.

The G/M transition
Entry into mitosis is controlled by the activation of cyclin B-CDK1 mitotic protein kinase
(96, 97). The cyclin B-CDK1 heterodimer also binds a small cyclin-kinase subunit (CKS) which
allows for binding to phosphoserine/phosphothreonine, helping the complex to maintain protein-
protein interactions allowing the complex to hyperphosphorylate its substrate by enabling
phosphorylation of even low affinity sites (99). This is important for the degradation of cyclin A
and the activation of the Anaphase Promoting Complex/Cyclosome (APC/C), an E3 ubiquitin
ligase. The mitotic cyclin-CDK complex accumulates in its inactive form, whereby it is
phosphorylated at a specific tyrosine residue and then is activated by dephosphorylation of the
inhibitory sites as previously described (98, 99). In mammalian cells, cyclin B1-CDK1 complexes
are activated over a period of 30 minutes which helps coordinate the onset of different events based
on dissimilar thresholds with cell rounding and nuclear import of cyclin B-CDK being the first
events initiated and APC/C and nuclear envelope breakdown (NEBD) being the last events (100).
There is some evidence that localization of cyclin B-CDK plays a role in this regulation as well,
first accumulating on centrosomes in human cells. Polo-like kinase 1 (PLK-1) also appears to play
a role in mitotic timing as inhibition of PLK-1 in human cells delays mitosis for several hours,
however the mechanism by which it controls mitosis is unclear, though it has been proposed to
promote rapid localization or accumulation of cyclin B1 to the nucleus before nuclear envelope
breakdown (100, 101).

24

Prophase
Prophase is defined by DNA condensation to form chromatids and breakdown of the
nuclear envelope. Sister chromatids (identical copies of replicated DNA) are bound together at the
centromere by cohesin, a multi-subunit protein complex comprised of two structural maintenance
of chromosomes (SMC) family proteins (SMC1 and SMC3), an alpha-kleisin, and a STAG
(stromal antigen) protein, to form chromosomes (102, 103). DNA condensation is a multi-level
reorganization of loose chromatin into tightly packed sister chromatids. This condensation
involves the wrapping of DNA around histones, specifically phosphorylated Histone H3, DNA
looping and DNA coiling (104). Several proteins aid in the organization and compaction of the
DNA including structural maintenance of chromosomes (SMC) protein complexes: cohesin,
condensin, and the SMC5-6 complex (105). Cohesin plays a dual role by participating in
chromosome condensation and sister chromatid cohesion. These ring-shaped structures are driven
by ATP hydrolysis and encircle strands or loops of DNA. Because of this compaction of DNA,
transcription is unable to occur (106). Additionally, the microtubules of the mitotic spindle are
formed at this time at the centrosomes which have migrated to opposite sides of the cell, hereby
referred to as spindle poles, via motor proteins (107).

Prometaphase, the Spindle Assembly Checkpoint, and the Mitotic Checkpoint Complex
In early prometaphase (which some still consider to be part of prophase), the nuclear
envelop is broken down by the phosphorylation of nuclear lamins and the mitotic spindle enters
the nuclear space. This type of mitosis where the nuclear envelope is dissolved so that microtubules
can invade the nuclear space is called open mitosis (99, 108).
25

During late prometaphase, the microtubules begin to attach to the centromeres of sister
chromatids via the kinetochore, the microtubule binding structure that is located atop the
centromere (109). The centromere is a specialized sequence of DNA that links sister chromatids
together. The microtubules that attach to the kinetochores are denoted as kinetochore microtubules
(kMT or K fibers). These microtubules interact with kinesin motor proteins in the kinetochore to
bind the microtubule to the chromosome (107). These interactions are initially rather unstable and
dynamic as interaction occurs in a lateral orientation, allowing the microtubules to grow and shrink
until a stable, end-on orientation can be achieved (110). There are 2 models of kinetochore
attachment to microtubules: the centrosome-mediated “search and capture” model and the
chromatin-mediated “self-organization” model (111-113). In the “search and capture” model,
microtubules originating from the centrosomes grow and shrink to probe the nuclear space until
they come in contact with kinetochores by chance. In the “self-organization” model, microtubules
nucleate around the chromatin eventually binding to the kinetochore by chance and spontaneously
assembling into antiparallel bundles forming microtubules connecting to the centrosomes.
Additionally, microtubules from one spindle pole will find and interdigitate with microtubules
from the opposite spindle pole referred to as polar microtubules. Between this point and
metaphase, we get activation of the last cell cycle checkpoint, the spindle assembly checkpoint
(SAC, also referred to as the spindle checkpoint, metaphase checkpoint, or mitotic checkpoint)
(114-116). The spindle assembly checkpoint halts mitotic progression until all sister chromatids
are attached to kinetochores emanating from opposite spindle poles (bipolar orientation/bi-
orientation, also known as amphitelic attachment), ensuring accurate separation of DNA into the
nascent daughter cells. Once all kinetochores are stably attached, the checkpoint is turned off and
the cells is allowed to progress through mitosis.
26

The spindle assembly checkpoint is controlled by a protein complex known as the mitotic
checkpoint complex (MCC). This multimeric protein complex is comprised of several proteins,
including BUB3, MAD2, BUBR1, and CDC20 (117). The formation of this complex is believed
to be initiated by the protein BUB1, often referred to as the “master regulator of the MCC,” but
Aurora B is also believed to play a role in recruiting MCC proteins to the kinetochore (118-122).
While there are several theories and models for MCC formation (reviewed in (123)), the most
accepted theory is the “MAD2 template model,” though details of this model are still not fully
understood (123). MAD2 exists in an inactive “open” conformation (O-MAD2) or an active
“closed” conformation (C-MAD2). The closed conformation creates a “safety belt” that allows
binding of MAD1 and CDC20. It had been proposed that MAD2 is synthesized in the open
conformation likely due to the barrier of activation energy making this conformation more
energetically favorable. It is thought that MAD2 may switch spontaneously from open to closed
as the closed confirmation is actually more stable. What causes conversion from the open to closed
confirmation is still unclear, but it has been suggested that formation of MAD1:C-MAD2 tetramer
may aid in the conversion of additional O-MAD2 to C-MAD2 where it can bind to other MAD1
proteins amplifying this confirmation. Increased C-MAD2 promotes its interaction with CDC20
and BUR1:BUB3, which is formed in a cell cycle independent manner, to form the MCC. In the
presence of kinetochores that are unattached to microtubules, the MCC is formed, generating a
“wait to enter anaphase” signal (124). Part of the function of the MCC is to bind to and sequester
CDC20, the activating component of a protein complex that promotes progression into anaphase,
the APC/C (117, 125). In this state, the APC/C cannot fulfill its role as a ubiquitin ligase. One of
the target proteins of the APC/C’s ubiquitin ligase function is securin (102). When the APC/C is
inactive, securin binds and inhibits separase, a protease that is responsible for cleaving cohesin,
27

halting the progression of mitosis until the criteria for mitotic progression is met, including
formation of the mitotic spindles/microtubules, attachment of microtubules to the kinetochore of
sister chromatids, generation of tension by movement of chromatids to opposing spindle poles,
and MAD2 stripping (115). The APC/C also targets cyclin B for degradation which triggers
anaphase progression (126).
Importantly, the kinetochores of sister chromatids must be attached to microtubules
emanating from opposite spindle poles. However, as microtubules attach, erroneous forms of
microtubule attachment can occur including syntelic and merotelic attachment (127). In syntelic
attachment, microtubules from the same spindle pole attach to both sister chromatids. In this
situation, the spindle assemble checkpoint remains active as this type of attachment does not
generate opposing tension on the sister chromatids and the MCC remains intact. In merotelic
attachment, a single kinetochore is attached to microtubules from both spindle poles. This form of
attachment is problematic as it still meets the conditions necessary to overcome the SAC.
Fortunately, the cell can survey for problematic attachments like this and correct them. The
chromosomal passenger complex (CPC), which is comprised of the kinase Aurora B and 3
regulatory and targeting components INCENP (Inner Centromere Protein), Borealin (also known
as Dasra) and Survivin (128). The CPC destabilizes these types of erroneous attachments by
phosphorylating the Ndc80 component of outer kinetochore which is involved with microtubule
binding which weakens its interaction with tubulin of microtubules allowing opportunity for the
correct bioriented attachments to occur (129, 130).

28

Metaphase
Once a microtubule attaches to the kinetochore of each sister chromatid, motor proteins
generate tension as they try to pull each of the sister chromatids to opposite spindle poles, causing
the chromosomes to align at the cells equator forming the metaphase plate defining metaphase
(131). This causes the MCC to dissociate, specifically MAD2 is moved away from the kinetochore
by dynein-dynein motor complex proteins which move MAD2 along the microtubule, in an event
referred to as MAD2 striping, releasing CDC20 and allowing it to activate the APC/C
(ACP/C
CDC20
) (132). Additionally, cyclin B1-CDK1 phosphorylates the APC/C, enhancing the
binding of CDC20 (102). Cyclin B1-CDK1 has also been known to phosphorylate CDC20,
however, the effect is unclear with conflicting evidence showing that phosphorylation can be
activating or inhibitory (133). The APC/C then acts as a ubiquitin ligase and targets the protein
securin for degradation, releasing separase where is the free to cleave cohesin (102). Cohesin is
cleaved at its kleisin subunit (134). At this point the SAC is deactivated, signaling that it is ready
to progress to the next stage of mitosis.
Once the cohesin ring is opened, the sister chromatids separate, allowing them freedom of
movement towards the spindle poles to which they are attached. Additionally, cyclin B1, the
mitotic cyclin, is targeted for degradation, helping promote progression towards anaphase and
mitotic exit (135). At this point, the APC/C targets polo-like kinase 1 (PLK1) and the Aurora
kinases for degradation and dephosphorylation of key proteins by protein phosphatase 1 (PP1) and
protein phosphatase 2a (PP2A) takes place (136, 137).
The APC/C actually has 2 activating co-factors. In addition to CDC20, CDH1 is also an
activator of the APC/C (136). This switch from CDC20 to CDH1 changes the conformation of the
APC/C changing the specificity of its ubiquitin ligase activity (138-140). The APC/C
CDH1
is
29

required for the exit of mitosis. In early mitosis, CDH1 is inactivated by phosphorylation by CDKs
and is then activated by dephosphorylation by CDC14 allowing it to bind to the APC/C. As an
active E3 ubiquitin ligase, the APC/C
CDH1
promotes mitotic exit by preventing the accumulation
of mitotic cyclins and other mitotic proteins that stabilize the cells entrance into G1, specifically,
APC/C
CDH1
targets cyclin A, cyclin B, Aurora B kinase, PLK-1, and CDC20 for ubiquitination and
thus degradation (141, 142)

Anaphase
The next phase of mitosis, anaphase, is broken down into 2 stages, anaphase A and
anaphase B (143-145). In anaphase A, the kinetochore microtubules begin to shorten, pulling the
newly separated chromatids towards the spindle poles in a process known as chromosome
segregation (143). During anaphase B, polar microtubules, which are microtubules emanating
from the spindle poles which interdigitate at the spindle midzone (the equator of the cell), push
against each other, causing the cell to elongate (144). At this point, the chromosomes further
condense to aid in the eventual reformation of the nucleus (146). The most important consequence
of anaphase is the equal separation of chromosomes into the daughter cells. Errors in anaphase can
cause changes or errors in DNA content that is inherited by the daughter cells. Specifically,
uncorrected merotelic attachments can cause lagging chromosomes that may interfere with proper
cell separation. Additionally, if the chromatids do not properly separate before the cells divide, the
cell may inherit an extra chromosome resulting in aneuploidy. Overall, defects during anaphase
can create inherited genetic instability (57, 127, 147, 148)

30

Telophase and Cytokinesis
The final stage of mitosis is telophase. At this point, the nuclear envelope reforms around
the separated chromatids and the cell continues to elongate. As the nuclear envelope reforms, the
DNA can begin to decondense as the cell exits mitosis.
In animal cells, mitosis concludes with cytokinesis. As telophase is occurring, the mother
cell abscises forming 2 daughter cells in a process known as cytokinesis. The process of cytokinesis
has several key steps: anaphase spindle reorganization, division plane specification, actin-myosin
ring assembly and contraction, and abscission (149).
During anaphase spindle reorganization, the mitotic spindle is reorganized to form the
central spindle (or spindle midzone) (112, 150-155). In this process, the mitotic spindle is bundled
together between the poles (156). Importantly, the central spindle plays a key role in regulating
cytokinesis as it coordinates recruitment of proteins involved in cleavage furrow positioning and
membrane abscission (128, 157, 158). The process of mitotic spindle reorganization in triggered
by the decrease in CDK1 activity (149). Loss of CDK1 activity causes loss of phosphorylation of
a CPC component, causing its translocation from the centromeres to the central spindle where it
plays a role in the phosphoregulation of other central spindle components including PRC1 (a
protein involved in microtubule bundling) and MKLP1 (a kinesin motor protein that binds to the
interface of antiparallel microtubules facilitating the spatial organization of the central spindle
microtubules). MKLP1 interacts with the Rho-family GTPase activating protein MgcRacGAP
(also known as CYK-4) forming the centralspindlin complex which in turn binds to the central
spindle as higher-order clusters which is regulated by Aurora A phosphorylation of MLKP1.
The specification of the site of the division plane is a topic of great debate. Currently, there
are 3 hypotheses addressing how the cell determines the plane of division for cleavage furrow
31

induction: the astral stimulation hypothesis, the central spindle hypothesis, and the astral relaxation
hypothesis (149). The astral stimulation hypothesis suggests that astral microtubules at the spindle
poles carry a furrow inducing signal to the cell cortex which is strongest at the central spindle. The
central spindle hypothesis suggests that the cleavage inducing signal comes from the central
spindle equator. The astral relaxation hypothesis suggests that there are active actin-myosin
bundles throughout the cell cortex and an inhibitory signal originating from the spindle poles is
weakest around the equator.
The cleavage furrow ingression is driven by the actin-myosin contractile ring located at the
equator of the cell cortex (149). Actin forms microfilaments while myosin is a motor protein that
moves along the actin filaments driven by ATP hydrolyses causing contraction thus partitioning
the cytoplasm of the emerging daughter cells.
The final stage of cytokinesis, abscission, is coordinated by the midbody structure (149).
While the midbody structure, which contains the bundled central spindle and other proteins, is
essential for cytokinesis, its components and mechanism is not fully understood. Just before
abscission, the two daughter cells are still tethered together by the intercellular bridge, where the
midbody structure is located. The abscission process is mediated by the ESCRT (endosomal
sorting complexes required for transport) machinery (159, 160). The recruitment of ESCRT
machinery to the midbody is initiated by centrosomal protein CEP55 which binds and MKLP1
(mitotic kinesin-like protein) and both are recruited to the midbody structure (153, 161). CEP55
then recruits ESCRT-I via its Vps23 subunit and ALIX, an accessory protein, to the midbody
structure which form rings on either side of the midbody (162). ESCRT-I and ALIX then recruit
ESCRT-III which forms spiral-like structure between the rings deforming the membrane where
the AAA-ATPase spastin is recruited by an ESCRT-III subunit to severe the central spindle (163-
32

165). The ESCRT machinery is then disassembled resulting in 2 newly formed daughter cells. The
new cells may now enter G0, resuming normal metabolic activity until the next round of
replication.

Rationale
As demonstrated, mitosis is a very complicated system that must be carried out precisely
in order to ensure proper genomic inheritance of the daughter cells. Because of this intricate
machinery, any errors in this chain of events can have disastrous consequences that can contribute
to disease development. Because ovarian cancer often presents as near tetraploid, it is likely that
this is a consequence of mitotic defects. The goal of this research is to understand mechanisms that
may contribute to or inhibit aneuploidy development as it pertains to disease development. As was
determined by previous students, our cell model undergoes a prolonged mitotic arrest during the
SAC, however the cause of the arrest was not known. The research presented here sought to further
elucidate the cause of this arrest. Using our cell model, we explored how sustained proliferation in
the background of deficient P53 can lead to aneuploidy development. We had previously published
that loss of BRCA1 lead to cells being able to overcome this mitotic arrest, but the mechanism by
which BRCA1 loss enables this was not known when I started this thesis project. My studies are
based on a hypothesis whereby cell-autonomous and cell-nonautonomous consequences of a
germline BRCA1 mutation cooperate with each other to promote ovarian cancer and seek to clarify
the cell-autonomous contributions. Lastly, a major goal of cancer research is to propose potential
treatments for these diseases with reduced negative side effects. Research by our lab and others
have proposed potential therapeutic value of inhibition of Aurora A. However, since Aurora A is
necessary for normal cellular function, we must understand the consequences of Aurora A
inhibition on mitotic regulation. I therefore sought to explore the potential merit of suppressing
33

Aurora A, a protein often overexpressed in cancer, in preventing tetraploid development and the
potential mechanism by which this could be achieved.
34

CHAPTER 2: MECHANISM OF CYTOKINESIS FAILURE IN OVARIAN
CYSTADENOMAS WITH DEFECTIVE BRCA1 AND P53 PATHWAYS

*Note: This work is published in the International Journal of Cancer, PMID: 29978915 (166)

INTRODUCTION

Mitosis is a highly orchestrated process regulated by multiple checkpoints. The spindle
assembly checkpoint, which takes place during prometaphase, ensures proper chromosome
segregation by preventing progression from metaphase to anaphase until each of the following is
completed: (i) spindle/microtubule formation, (ii) attachment of the microtubules to the
kinetochore, (iii) generation of tension on the centromeres of sister chromatids by motor proteins,
and (iv) dissociation of the mitotic checkpoint complex (115). This multiprotein complex, present
at the kinetochores of sister chromatids, sequesters CDC20, the activating component of the
anaphase promoting complex (117). Premature exit from this checkpoint can lead to cytokinesis
failure due to the presence of lagging chromosomes or abnormal spindle anchoring (56). This
results in polyploidy and subsequent aneuploidy, one of the hallmarks of cancer. Several cancer
types, particularly those associated with the BRCA1 mutation carrier state, are characterized by an
aneuploid state that is near polyploid, suggesting a role for this scenario in their development (55).
Women harboring germline mutations in BRCA1 or BRCA2 are predisposed to triple
negative breast carcinomas and high-grade serous carcinomas in the ovary, which will be referred
to as high-grade serous extra-uterine Müllerian carcinomas in this manuscript because, as
explained earlier, this nomenclature more accurately conveys the true site of origin of these cancers
(15). Indeed, the notion that these tumors originate in the ovarian surface epithelium has been
35

challenged by the scientific community (167, 168). Numerical chromosomal abnormalities
characterized by a near polyploid aneuploid state, together with the presence of a P53 mutation,
are the only clonal abnormalities commonly associated with these cancers, hence the idea that they
are primarily a disease of chromosomes as opposed to being driven by alterations in specific
metabolic pathways (169). We previously developed an in vitro cell culture model where serous
extra-uterine Müllerian cystadenomas, which are benign tumors of the same differentiation lineage
as high-grade serous extra-uterine Müllerian carcinomas, were transfected with SV40 large T
antigen to extend their in vitro life span (59, 60). This antigen, by inhibiting the P53 and RB
proteins, mimics alterations typically present in early cancers associated with the BRCA1 mutation
carrier state. Indeed, clonal P53 mutations are regarded as a molecular signature for cancer
precursor lesions in BRCA1 mutation carriers in the absence of any morphological evidence of
cancer (170). Forced expression of SV40 large T antigen was also successfully used in cell culture
and transgenic models of high-grade serous extra-uterine Müllerian carcinomas in other
laboratories (171, 172).
We reported earlier that this in vitro culture model is characterized by a cell cycle arrest at
the spindle assembly checkpoint as the cells reach high replicative ages in vitro, which can be
overcome by down-regulation of BRCA1 to levels comparable to those present in human BRCA1
mutation carriers (173). Such premature recovery from the spindle assembly checkpoint is not
without cost, as it can lead to cytokinesis failure resulting in polyploidy and subsequent
aneuploidy. An association between decreased BRCA1 levels and risk of polyploidy was also
reported by Wang et al. (174) in a slightly different in vitro model. This chain of events can lead
to spontaneous malignant transformation and, in fact, led to the establishment of immortal cell
lines in our laboratory (60). We hypothesize, based on observations not only with this in vitro
36

model, but also with mice carrying a conditional Brca1 knockout (175) as well as observations
with human populations (50, 51), that the molecular events mediating predisposition to triple
negative breast carcinoma and high-grade serous extra-uterine Müllerian carcinoma in BRCA1
mutation carriers entail an interplay between cell-nonautonomous consequences of this carrier
state resulting in premature replicative aging due to increased cellular proliferation, and cell-
autonomous consequences leading to numerical chromosomal abnormalities due to cytokinesis
failure that are especially frequent in cells of advanced replicative ages (173).
We sought to better understand the mechanism underlying the prolonged arrest at the
spindle assembly checkpoint associated with increased replicative age, which is a central element
of this proposed interplay between cell-autonomous and -nonautonomous mechanisms of cancer
predisposition, in an effort to gain insights into the molecular events associated with early cancer
development in individuals with germline BRCA1 mutations.

MATERIALS AND METHODS

Cell strains and culture conditions
The isolation and characterization of ML10 cells was previously described (59). ML10 cells were
grown in Minimal Essential Medium (MEM) supplemented with 10% Fetal Bovine Serum (FBS)
and 50U Penicillin, 50 µg Streptomycin per ml. Cells were incubated at 37
o
C under atmospheric
conditions containing 95% humidity and 5% carbon dioxide.

37

Confocal immunofluorescence imaging
Cells were cultured on LabTek II cell culture chamber slides (VWR, Cat# 62407-290). The slides
were placed in phosphate buffered saline (PBS) containing 4% paraformaldehyde and 0.2% Triton
X-100 for 30 minutes at 37
o
C for visualization of nuclear proteins, or at room temperature for
visualization of total cellular proteins, followed by three 5-minute washes in PBS and incubation
for 1 hour at room temperature in PBS containing 1% bovine serum albumin used as blocking
agent. For studies entailing imaging of DNA only, the cells were mounted in SlowFade Gold
Antifade Mountant with DAPI (ThermoFisher Scientific, Cat# S36938) and cover slipped. For
protein stains, the cells were probed overnight with primary antibody, washed 3 times for 5 minutes
with PBS, hybridized to appropriate fluorescent-labelled secondary antibodies for 2 hours at room
temperature in the absence of light, and washed 3 times for 5 minutes. The slides were cover
slipped and visualized using a PerkinElmer Spinning Disk Confocal Microscope with 63X
objective and captured and analyzed using the Volocity system.

Protein extraction procedures
For nuclear and cytoplasmic protein separation and extraction, tissue culture dishes were briefly
rinsed with ice cold PBS, lysed in Buffer A [0.1% Triton-X 100, 20mM N-ethylmaleimide (NEM),
10mM HEPES pH 7.9, 10mM potassium chloride, 10mM beta-glycerophosphate, 1mM sodium
vanadate, 0.5mM phenylmethanesulfonyl fluoride (PMSF), 2ug/ml aprotinin, 1.5mM magnesium
chloride, 1ug/ml leupeptin, 1ug/ml pepstatin A, and 0.5mM dithiothreitol (DTT)], and harvested
from the dish bottom by scraping. The cell lysates were rocked for 20 minutes at 4
o
C and
centrifuged at 1150 x g for 10 min at the same temperature. The supernatant containing the
cytoplasmic proteins was recovered and the cell pellet was resuspended in Buffer C [0.42mM
38

sodium chloride, 6.25% glycerol, 20mM NEM, 20mM HEPES pH 7.9, 1mM sodium vanadate,
10mM beta-glycerophosphate, 2ug/ml aprotinin, 1.5mM magnesium chloride, 1ug/ml leupeptin,
1ug/ml pepstatin A, 0.5mM DTT, 0.2mM ethylenediaminetetraacetic acid (EDTA), and 0.5mM
PMSF]. The resuspended pellets were rocked for 30 minutes at 4
o
C and centrifuged at 4
o
C for 30
min at 15871 x g. The supernatants containing the nuclear proteins were collected and stored
at -80
o
C until use.
For total protein extraction the cells were lysed in Triton lysis buffer (TLB pH 8.0,
containing 25mM sodium phosphate, 150 mM sodium chloride, 1% Triton X-100, 5mM EDTA
50mM sodium fluoride) freshly supplemented with 1mM PMSF, 1mM sodium vanadate, 10ug/ml
aprotinin, 10ug/ml leupeptin, 5uM pepstatin A, 25mM phenylarsine oxide (PAO), rocked for 30
minutes at 4
o
C and centrifuged at 4
o
C for 30 min at 15871 x g. The supernatants containing the
total proteins were collected and stored at -80
o
C until use.

Western blotting
Proteins larger than 100kDa and those between 37-100kDa were electrophoresed on 8% and 10%
polyacrylamide gels, respectively, while those smaller than 37kDa were separated on 12%
polyacrylamide gels. The protein samples were dissolved in 6X Laemmli buffer (0.416M sodium
dodecyl sulfate, 0.896mM Bromophenol Blue, 47%/6.44M glycerol, 85mM Tris pH 6.8, 0.6M
dithiothreitol), placed in boiling water baths for 5 min, loaded onto gels, and electrophoresed at
80V for 30 min, followed by 150V for 60-70 minutes. Wet transfer to PVDF membranes (BioRad,
Cat# 1620177) was performed at 40V for at least 24 h at 4
o
C. The membranes were briefly washed
with Tris buffered saline (TBS) (pH 8.1) containing 0.1% Tween 20 and blocked for 1 hour at
room temperature in the same buffer supplemented with 5% blocking grade milk proteins (BioRad,
39

Cat# 1706404). The membranes were probed overnight at 4
o
C with primary antibody diluted in
TBS-Tween20-blocking proteins, washed, and hybridized to secondary antibody for 1 hour at
room temperature followed by a final wash in TBS-Tween20-blocking proteins. The membranes
were treated with SuperSignal West Pico Chemiluminescent Substrate (ThermoFisher Scientific,
Cat# 34077) and exposed to X-ray films.

Time lapse microscopy
The cells were cultured on glass chamber slides and treated with siRNA against either GFP or
BRCA1 for 24 hours before being transferred to a Perkin Elmer Spinning Disk Confocal
microscope fitted with an incubator under controlled temperature and atmospheric conditions.
Photographs were taken every minute over a 20-hour period using the 10X objective and captured
and analyzed using the Volocity system.

Transfection with siRNA
The siRNA sequences used for BRCA1 down-regulation were published (173). The siRNA
sequence against Green Fluorescence Protein used as control was 5'-
GACGUAAACGGCCACAAGU-3'. Transfection was achieved using Lipofectamine 2000
Transfection Reagent (ThermoFisher Scientific, Cat# 11668019) according to manufacturer’s
protocol.

Source and dilutions of antibodies
For confocal microscopy, the following antibodies were used at a dilution of 1 µg/mL: EB1 (BD
BioScience, Cat# 610535) and BUB3 (Santa Cruz Biotechnology, Cat# sc-136217). Secondary
40

antibodies included goat anti-mouse IgG (H+L) Alexa Fluor 555 conjugate (Thermo Fisher, Cat#
A21422) and goat-anti-rabbit IgG (H+L). Alexa Fluor 647 conjugate (Thermo Fisher Cat# A-
21244) used at a dilution of 1µg/mL. For western blotting, dilutions of 1:1000 were used for
antibodies against Ku70 (Santa Cruz Biotechnology, Cat# sc-12729), GAPDH (Santa Cruz
Biotechnology, Cat# sc-25778), and BUB3, and dilutions of 1:500 were used for antibodies against
EB1, phospho-BUB3 (Thermo Fisher, Cat# PA5-37772), and BUB1 (Santa Cruz Biotechnology,
Cat# sc-28257).

Statistical analyses
We used 2-tailed Fisher’s exact test to compare proportions of the different parameters of interest
between different cell populations.

RESULTS

Mechanism of metaphase arrest in aging ovarian cystadenomas
We reported earlier that in vitro cultures of extra-uterine Müllerian serous cystadenomas, which
are of the same differentiation lineage as the cancers of the female reproductive tract that are
typically associated with the BRCA1 mutation carrier state, show increased predisposition to cell
cycle arrest at the spindle assembly checkpoint when they reach advanced replicative age (173).
We sought to better understand the mechanisms underlying this arrest by investigating changes in
the intracellular distribution of BUB3, a component of the metaphase checkpoint complex also
involved in proper microtubule attachments, as a function of replicative aging. Representative
examples of cells with predominantly nuclear and cytoplasmic distribution of BUB3 during
41

mitosis are shown in Fig. 2A. Eighteen of 22 (82%) mitotic cells with a replicative age of less than
35 population doublings (low replicative age) showed a primarily nuclear distribution of BUB3
compared to 7 of 36 (19%) mitotic cells with a replicative age of more than 50 population
doublings (high replicative age), an age at which most cells are arrested at the spindle assembly
checkpoint (Fig. 2B). The majority of the cells in the latter age group showed a predominant
cytoplasmic distribution of BUB3. This difference in the intracellular distribution of BUB3
between cells of low versus high replicative age is further supported by the demonstration of
increased cytoplasmic BUB3 in the absence of a corresponding increase in total cellular BUB3 in
aging populations by western blotting analyses (Fig. 2C). The presence of cytoplasmic BUB3 in
cells of low replicative age in these studies performed on heterogeneous cell populations not
synchronized in their cell cycle is explained by the fact that BUB3 is a multi-functional protein
not expressed solely during mitosis
BUB3 is a target of phosphorylation by the BUB1 kinase, known as the master regulator
of the metaphase checkpoint complex formation (176). The phosphorylated form of BUB3 recruits
microtubules to the kinetochores to promote anchoring (121). We hypothesized that failure of
BUB3, a mitotic checkpoint complex protein, to associate with chromosomes in cells of higher
replicative age, which can readily account for the observed prolonged mitotic arrest in this cell
population, might reflect a decrease in BUB3 phosphorylation. Indeed, a substantial relative
decrease in the phosphorylated form of BUB3 was seen by western blotting analyses in cell
populations with more than 50 population doublings compared to populations with less than 35
population doublings (Fig. 2C). This decrease coincided with a decrease in total cellular BUB1
(Fig. 2C).
42

Such decrease in BUB3 phosphorylation supports the idea that recruitment of microtubules
to the kinetochore is hindered in aging cell populations. We investigated this possibility further by
examining the intracellular distribution of End Binding Protein 1 (EB1), a protein that accumulates
at the kinetochores upon microtubule anchoring and stabilizes their attachment to centromeres
(177). Western blots probed with an antibody against EB1 showed decreased levels of this protein
in cells of high replicative age. These differences were most prominent in nuclear extracts
compared to whole cell extracts (Fig. 2D). This conclusion was supported by comparing the
intracellular localization of EB1 to that of nuclear BUB3 (used as surrogate marker of kinetochore
localization) in mitotic cells from the 2 age groups by confocal microscopy (Fig. 2E).
Quantification of the confocal observations showed that EB1 was not only primarily localized to
the DNA, but also showed strong colocalization with BUB3 in the younger age group as expected
while it was primarily localized to the centrosomes, the site of microtubule nucleation, in cells
from the older age group (Fig. 2E-F). We conclude that the mitotic arrest at the spindle assembly
checkpoint that characterizes aging serous ovarian cystadenomas is due, at least in part, to a defect
in microtubule anchoring.

Fate of arrested cells
Cohesion fatigue is a possible consequence of a prolonged mitotic arrest such as that associated
with replicative aging in our cell culture model. This phenomenon is manifested by asynchronous
migration of chromatids from the metaphase plate in cells that are arrested or delayed in metaphase
(178). It leads to either cell death or, less commonly, to mitotic slippage and cytokinesis failure,
as typically observed in aging populations of cultured ovarian cystadenomas (173). Fig. 3A shows
2 different cells examined by confocal microscopy after visualization of the metaphase plates using
43

DAPI staining: one (left) with an intact metaphase plate associated with a normal metaphase and
the other (right) illustrating migration of chromatids from the metaphase plate indicative of
cohesion fatigue. Fifty-seven percent (38 of 67) of mitotic cells with a replicative age higher than
50 population doublings showed evidence of cohesion fatigue when examined by confocal
microscopy compared to 18% (8 of 44) of mitotic of cells in the younger replicative age group
(Fig. 3B). We conclude that the increased rate of cell death and of cytokinesis failure associated
with a prolonged cell cycle arrest in our cell culture model are mediated, at least in part, by
cohesion fatigue.

Consequences of BRCA1 down-regulation
Premature recovery from the arrest at the spindle assembly checkpoint associated with replicative
aging in cultures of ovarian cystadenomas can be achieved by down-regulation of BRCA1, a
protein controlling familial predisposition to triple-negative breast and high-grade serous extra-
uterine Müllerian carcinomas (173). Given our current data suggesting a role for defective
microtubule anchoring in causing this arrest, we hypothesized that decreased BRCA1 expression
could somehow overcome this defect. Western blot analysis of nuclear protein extracts derived
from cultured serous cystadenomas showed increased EB1 levels following treatment with siRNA
against BRCA1 (siBRCA1) in support of this hypothesis (Fig. 3C). This conclusion was further
supported by confocal microscopy, which typically showed doubling of the size of EB1 signals
following down-regulation of BRCA1 (Fig. 3D). This was associated with a decrease in the
proportion of aging cells undergoing cohesion fatigue, as 38 of 67 (57%) cells treated with siRNA
against GFP (siGFP) showed this abnormality compared to 26 of 70 (37%) cells treated with
siBRCA1 (Fig. 3E). This is consistent with the notion that increased microtubule anchoring
44

triggered by BRCA1 down-regulation relieves the cells from the stress associated with a prolonged
mitotic arrest. Such down-regulation did not lead to a significant reduction in cohesion fatigue in
the younger cell population, as this mitotic abnormality was seen in 8 of 44 (18%) cells treated
with siGFP and in 10 out of 62 (16%) cells treated with siBRCA1 (Fig. 3E). This was expected
because prolonged arrests at the spindle assembly checkpoint due to defective microtubule
anchoring are infrequent in this population.
Our previous results showed that down-regulation of BRCA1 leads to a marked increase
in cytokinesis failure resulting in polyploidy (173). Dysregulated microtubule attachment can lead
to abnormal attachment to the kinetochore such as, for example, merotelic attachments, preventing
normal cytokinesis (147). Such attachments are seen when the kinetochore of a sister chromatid is
attached to microtubules from both spindle poles, generating opposing pulling forces. The
condensed chromatid strands unravel under such conditions as they are being pulled to opposite
poles resulting in chromatid strings or bridges between the two chromosome plates. Minor
bridging (2 or fewer bridges) is compatible with normal cytokinesis because it can be overcome
by the force of cleavage furrow ingression. More severe bridging (more than 2 bridges), however,
cannot be overcome by similar forces resulting in cytokinesis failure and the generation of
binucleated tetraploid cells (179). Examples of cells showing either no bridging, minor bridging,
or severe bridging in our cell culture model are shown in Fig. 4A. We hypothesized that the
resumption of mitotic progression associated with cytokinesis failure observed after BRCA1
down-regulation in aging cell populations might be accounted for by a chain of events triggered
by increased microtubule anchoring resulting in abnormal attachment to the kinetochore and
formation of bridges between spindle poles thus interfering with cleavage furrow ingression.
Severe bridging was rarely present in cells of low replicative age regardless of their state of
45

BRCA1 down-regulation (present in 2% of cells treated with siRNA against GFP compared to 4%
of cells treated with siRNA against BRCA1) (Fig. 4B). This is consistent with the fact that a
prolonged mitotic arrest due to defective microtubule anchoring is rarely present in this age group,
thus negating the effect of BRCA1 down-regulation. In contrast, the rate of severe bridging was
increased substantially in aging cells following treatment with siRNA against BRCA1 (53% of
cells treated with siRNA against BRCA1 versus 22% of controls) (Fig. 4B), likely due to
unsuccessful attempts at correcting anchoring deficiencies. We conclude that reduced BRCA1
levels lead to uncontrolled anchoring of microtubules to the kinetochore and subsequent
chromosome bridging and cytokinesis failure, accounting for the increased rate of polyploidy
reported earlier in aging cell populations following BRCA1 down-regulation (173). The fact that
22% of aging cells treated with siGFP showed severe bridging suggests that some degree of
abnormal microtubule attachment also occurs spontaneously in cells undergoing a prolonged
arrest.
We examined the mitotic fate of aging cells treated with either control siRNA (siGFP) or
siRNA directed against BRCA1 by time-lapse photography to further support our conclusions. All
cells that attempted telophase in these two treatment groups were examined by 3 independent
observers in a time-lapse video covering a period of 20 hours. Those that demonstrated cleavage
furrow ingression followed by complete cytoplasmic separation were categorized as achieving
complete cytokinesis. Cells that entered mitosis but did not resolve into 2 nascent daughter cells,
regardless of attempt at cleavage furrow ingression, were categorized as showing cytokinesis
failure. As shown in Fig. 5A, the rate of cytokinesis failure was significantly higher in cells treated
with siRNA targeted against BRCA1 (67.9%) compared to the control cells treated with siGFP
(19.8%) (P = 0.0001). This experiment was repeated a second time with a different movie and
46

showed similar results. Fig. 5B shows snapshots of a representative cell entering mitosis and
subsequently failing cytokinesis. The entire movie can be seen in Suppl. Fig. 1 of the published
paper (166).

47

DISCUSSION

Our results clearly show that sustained cellular proliferation leading to increased replicative
age in the absence of a normal P53 function impairs normal anchoring of microtubules to the
kinetochore in in vitro cultures of serous extra-uterine Müllerian cystadenomas undergoing
mitosis. This coincides with a decrease in expression of the BUB1 kinase, which is the master
regulator of metaphase checkpoint complex formation, and in a concomitant decrease in
phosphorylation of its substrate, BUB3. The ensuing prolonged mitotic arrest eventually leads to
uncoordinated migration of chromosomes to the mitotic spindle poles due to cohesion fatigue.
Although cell death is the most likely outcome, mitotic slippage occasionally leads cells to survive
at the expense of cytokinesis failure and polyploidy due to interference of lagging chromosomes
with cleavage furrow ingression. The likelihood of cytokinesis failure is magnified if BRCA1
expression is decreased to levels approximating those present in human BRCA1 mutation carriers
because such reduction leads to increased and uncoordinated attachments of microtubules to the
kinetochore. This results not only in sufficient tension to overcome the aforementioned failure of
microtubule anchoring, but also in abnormal attachments to the kinetochore, such as merotelic
attachments, which are defined as anchoring of microtubules from opposite poles to the same
kinetochore. Evidence for increased occurrence of such attachments in cells with decreased
BRCA1 levels comes from higher incidence of chromatid bridges between chromosomal plates,
indicating the presence of forces pulling individual chromosomes in opposite directions. This
results in cytokinesis failure due to interference with cleavage furrow ingression.
Aneuploidy associated with a near polyploid state, which is a consequence of reduced
BRCA1 expression (173), is often the only genetic abnormality present in high-grade serous extra-
48

uterine Müllerian carcinomas in addition to abnormalities in P53 (169). Our findings shed light
not only on the molecular events predisposing to aneuploidy, but also on mechanisms that magnify
its frequency in BRCA1 mutation carriers. The results also provide a possible explanation for the
restricted site specificity of the cancers associated with the BRCA1 mutation carrier state, which
are limited to the breast and to the reproductive tract in spite of the fact that BRCA1 is widely
expressed in most tissues. Indeed, our data suggest that these consequences of reduced BRCA1
expression on premature progression to anaphase resulting in cytokinesis failure are only
significant in cells subjected to accelerated replicative aging, a condition that can be triggered in
mammary and in serous extra-uterine Müllerian epithelia due to cell-nonautonomous
consequences of the BRCA1 mutation carrier state (46, 48-50). Evidence from observations with
animal models as well as with human studies (46, 48-50) suggest that the BRCA1 mutation carrier
state leads to changes in the hormonal fluctuations associated with the menstrual cycle, which are
the most important risk factor for the sporadic (non-familial) forms high-grade serous extra-uterine
Müllerian and breast carcinomas (10, 180). Such hormonal fluctuations can influence mitogenic
pathways such as, for example, the RANK pathway in targeted tissues (51, 181), resulting in
premature replicative aging due to increased cellular proliferation. Our working hypothesis for
high-grade serous extra-uterine Müllerian and triple negative breast carcinoma predisposition
based in this scenario is summarized in Fig. 6. This model readily accounts for the well-established
notion that interruption of the menstrual cycle via either pregnancy or oral contraceptive use has
profound protective effects against high-grade serous extra-uterine Müllerian and breast
carcinomas not only in the general population (10, 180), but also in BRCA1 mutation carriers (8,
9, 13).
49

The molecular events leading to cytokinesis failure and polyploidy associated with early
cancer development in BRCA1 mutation carriers most likely continue to operate after malignant
transformation and undoubtedly influence the biology of these cancers. For example, they may
contribute to the generation of polyploid giant cancer cells, recently shown in Jinsong Liu’s
laboratory (182, 183) to express normal and cancer stem cell markers and to have the ability to
increase their viability by reducing their DNA content via asymmetric divisions, budding, or
bursting. This group suggested that these events, which are related to an early embryological
mechanism common in the blastomere stage of preimplantation embryos, may represent important
mechanisms for generating cancer stem-like cells and may also contribute to tumor heterogeneity
(184).
The exact mechanisms whereby decreased BRCA1 expression can lead to uncontrolled
anchoring of microtubules to the kinetochore are still unclear. The consequences of these
abnormalities on the normal events needed to ensure proper homeostasis during mitotic
progression are also incompletely understood. BRCA1 acts as a ubiquitin ligase when bound to
BARD1 (185), causing degradation of gamma-tubulin, the nucleating component for microtubule
polymerization. Loss of this function leads to an increase in microtubule nucleation as
demonstrated by increased formation of astral microtubules (31). It is possible that reduced
availability of BRCA1 interferes with the control of this process, leading to excessive and poorly
regulated microtubule anchoring. Another possibility is that the Chromosomal Passenger
Complex, which is involved in regulating chromosome bi-orientation in metaphase, requires an
intact BRCA1 function. Aurora B, a component of this complex, might be especially affected by
the dysregulation in microtubule anchoring associated with deficiencies in BRCA1, as one of its
functions includes correction of abnormal attachments to the kinetochores (186).
50

A better understanding of the mitotic events triggered by BRCA1 deficiencies might lead
to the identification of biomarkers associated with these events that could be evaluated in biopsies
of breast or fallopian tube before malignant transformation and provide important tools in the
clinical management and follow up of BRCA1 mutation carriers. For example, they could be used
as intermediate surrogate biomarkers of response in cancer risk reduction trials. This knowledge
could also facilitate the development of non-surgical means of cancer risk reduction in BRCA1
mutation carriers based on targeting mediators of the events leading to mitotic errors. The only
effective means of cancer risk reduction in BRCA1 mutation carriers currently entails surgical
removal of both breasts and reproductive organs, with important consequences including
infertility, premature menopause, and social/emotional side effects (12). This leads to many
individuals to postpone their surgery and thus increase their risk of cancer, underscoring the need
for such non-surgical approaches to cancer risk reduction in this population. Agents targeting Poly
(ADP ribose) Polymerase (PARP) proteins might have merit in this context, as PARP inhibition
appears to recapitulate phenotypic features associated with replicative aging observed in our
present study, namely pre-anaphase arrest, cytokinesis failure, and multinucleation. Indeed, there
is increasing evidence supporting a role for poly(ADP ribosyl)ation of BUB3 and other
centromeric proteins in mitotic progression, including for proper spindle assembly and function
(23, 187-192). While PARP inhibitors are often used for the treatment of the cancers developing
in the context of the BRCA1 mutation carrier state given the dependence of these cancers on PARP
proteins for DNA repair functions (22), their potential effects on cancer predisposition due to
interference with the role of PARP proteins in mediating the mitotic events influenced by this
carrier state, such as those reported here, merit further investigation. In addition, the idea that
PARP inhibitors are effective in the treatment of BRCA1-deficient cancers due primarily to
51

accumulation of double stranded DNA breaks secondary to deficient homologous recombination
has been challenged (21). Thus, exploring the function of PARP proteins in the regulation of the
mitotic events altered in BRCA1-deficient cells including those addressed in our study may
provide important insights into the mechanism through which PARP inhibition induces cell death
in such cancers.

52

Figure 1: Reduced microtubule anchoring in aging ovarian cystadenomas

53

Figure 2: Reduced microtubule anchoring in aging ovarian cystadenomas. A: Confocal images of
ovarian cystadenomas in prometaphase/metaphase stained with a fluorescent antibody against
BUB3 (red) and counterstained with DAPI (blue) showing representative examples of
predominantly nuclear (left) and cytoplasmic (right) distribution of BUB3. B: Cystadenomas of
low (<35 population doublings) versus high (>50 population doublings) replicative ages were
examined by confocal microscopy as in (A); mitotic cells were scored based on presence of
predominantly nuclear versus cytoplasmic distribution of BUB3; the numbers on the stacked bars
represent the total number of cells for each parameter. C: Western blots of either whole cellular
protein extracts or of proteins extracted from the cytoplasmic fraction only of cultured
cystadenomas with less than 35 versus more than 50 population doublings were probed with
antibodies against the indicated proteins; GAPDH was used as loading control. D: Western blots
of nuclear and whole cell protein extracts of cystadenomas of low and high replicative ages probed
with an antibody against EB1; Ku70 and GAPDH were used as loading controls for nuclear and
cytoplasmic proteins, respectively. E: Confocal microscopy images showing examples of ovarian
cystadenomas undergoing mitosis with and without EB1 localization to the kinetochore;
immunopositivity for BUB3 was used as a surrogate marker for the kinetochore. F: Cystadenomas
of low (<35 population doublings) versus high (>50 population doublings) replicative ages were
examined by confocal microscopy as in (E) and the mitotic cells were scored based on presence
versus absence of colocalization of EB1 and BUB3 to the kinetochore; the numbers on the stacked
bars represent the total number of cells for each parameter. Magnification bars: 6 microns.

54

Figure 2:Mechanism of escape from the spindle assembly checkpoint in cells with reduced BRCA1 levels
55

Figure 3: Mechanism of escape from the spindle assembly checkpoint in cells with reduced
BRCA1 levels. A: Confocal images of cells stained with DAPI showing a normal metaphase (left)
and a metaphase undergoing cohesion fatigue (right). B: Cystadenomas of low (<35 population
doublings) versus high (>50 population doublings) replicative ages were examined by confocal
microscopy and the mitotic cells were scored based on presence of either normal metaphases or
metaphases showing evidence of cohesion fatigue; the numbers on the stacked bars represent the
total number of cells for each parameter. C: Western blot of nuclear or total protein extracts
obtained from cystadenomas treated with siRNA directed against either BRCA1 or GFP (control)
and probed with an antibody against EB1; a probe for Ku70 was used to evaluate loading of nuclear
proteins while a probe for GAPDH was used to evaluate loading of cytoplasmic protein extracts.
D: Cells stained with an antibody against EB1 (green) and counterstained with DAPI (blue) were
examined by confocal microscopy and photographed under constant exposure; these representative
images illustrate the increase in the size of EB1 signals in cells treated with siRNA directed against
BRCA1 compared to cells treated with siRNA directed against GFP. E: Cystadenomas of low (<35
population doublings) versus high (>50 population doublings) replicative ages were treated with
siRNA directed against either BRCA1 or GFP and examined by confocal microscopy; the mitotic
cells were scored based on presence of either normal metaphases or metaphases showing evidence
of cohesion fatigue; the numbers on the stacked bars represent the total number of cells for each
parameter. Magnification bars: 6 microns in (A) and 8 microns in (D).

56

Figure 3: Consequences of BRCA1 down-regulation on formation of bridges between chromosome plates
Figure 4: Consequences of BRCA1 down-regulation on formation of bridges between
chromosome plates. A: Examples of mitotic cells in anaphase showing varying degrees of
chromosome bridging seen by confocal microscopy after staining with DAPI; magnification bars:
8 microns. B: Cystadenomas of low (<35 population doublings) versus high (>50 population
doublings) replicative ages were examined by confocal microscopy as in (A); cells undergoing
anaphase were scored based on presence of either absent/minor or severe chromosome bridging;
the numbers on the stacked bars represent the total number of cells for each parameter.

57

Figure 4: Consequences of BRCA1 down-regulation on cytokinesis failure
*This figure was done in collaboration with Dr. Vanessa Yu and Dr. Christine Marion
Figure 5: Consequences of BRCA1 down-regulation on cytokinesis failure. A: Cystadenomas of
high replicative ages were treated with siRNA directed against either BRCA1 or GFP and
examined by time-lapse photography over a 20-hour period; all cells undergoing mitosis were
followed and scored as showing either completion of cytokinesis or cytokinesis failure; the
numbers on the stacked bars represent averages of the total number of cells for each parameter;
the error bars represent variation in independent observations of the entire 20-hour video by 3
observers. Selected frames from the time-lapse microscopy video used are shown in panel (B),
illustrating a mononuclear cell subsequently undergoing a mitotic arrest followed by cytokinesis
failure resulting in a multi-nucleated interphase cell. The entire video is submitted as supplemental
data in published paper (166).
58

Figure 5: Working model for interplay between cell-nonautonomous and cell-autonomous mechanisms of cancer predisposition
in BRCA1 mutation carriers
59

Figure 6: Working model for interplay between cell-nonautonomous and cell-autonomous
mechanisms of cancer predisposition in BRCA1 mutation carriers. The menstrual cycle, under the
influence of ovarian granulosa cells and cells from the anterior pituitary, control the proliferation
of tissues that have an elevated cancer risk in BRCA1 mutation carriers (170). These cell-
nonautonomous signals are amplified in such carriers resulting in accelerated replicative aging.
P53 alterations, which are frequent in these tissues in BRCA1 mutation carriers, eventually lead to
mitotic arrest in aging cells due to defective microtubule anchoring. Cohesion fatigue is the most
likely outcome in tissues with premature replicative aging due to increased menstrual cycle activity
in the absence of a germline BRCA1 mutation, but some cells abruptly exit the arrest due to mitotic
slippage resulting in cytokinesis failure and binucleation due to interference of lagging
chromosomes or microtubules at the site of cell cleavage. The presence of a germline BRCA1
mutation not only intensifies accelerated replicative aging, but also leads to recovery from the
mitotic arrest due to uncontrolled kinetochore attachments resulting in the formation of
chromosome bridges; this significantly increases the rate of cytokinesis failure, leading to
polyploidy followed by aneuploidy and malignant transformation.

60

CHAPTER 3: AURORA A KINASE REGULATES CHROMOSOME CONDENSATION
WITH CONSEQUENCES FOR METAPHASE TO ANAPHASE TRANSITION

INTRODUCTION
Aneuploidy contributes to biological aggressiveness, tumor malignancy, and poor
prognosis in ovarian cancer (52, 53, 193). It is also believed that aneuploidy development is an
early event is ovarian cancer tumorigenesis (194). Due to the prevalence of aneuploidy in high-
grade serous ovarian carcinomas, ovarian cancers are considered a disease of chromosomes (169).
Aneuploidy can lead to genetic variation within a tumor contributing to difficulties in treatment,
such as the development of chemoresistant cells (195). Thus, prevention of aneuploidy
development is essential to inhibiting carcinogenesis, tumor evolution, and heterogeneity. Many
chemotherapeutic agents work by targeting rapidly dividing cells by negatively affecting the cell
cycle. For instance, platinum-based drugs such as carboplatin and cisplatin, which are often
prescribed for ovarian cancer treatment, interfere with DNA replication (16). Taxanes, including
paclitaxel and docetaxel, are microtubule toxins that prevent microtubule depolymerization
preventing mitotic progression (196). However, these agents work rather indiscriminately
affecting both cancerous cells and rapidly growing healthy cells. Due to this lack of specificity,
efforts have been focused on achieving a more targeted effect. The approach to this goal is to
exploit molecular differences between cancerous and normal cells such as protein overexpression.
The kinase Aurora A belongs to a family of serine/threonine kinases that play varying and
crucial roles in mitotic regulation, including Aurora B and Aurora C. Aurora A has been shown
to be involved in centrosome maturation, centrosome separation, and bipolar spindle formation
(197). Studies have shown that Aurora A may be involved in cytokinesis, as it localizes to the
61

midbody structure during cytokinesis, though it is unclear how Aurora A in involved (198). More
recently, Aurora A has been implicated in tumorigenesis in a variety of cancer types and is thought
to be an oncogene when overexpressed and has been found to be overexpressed in a variety of
cancers (199). Overexpression of the kinase is associated with aneuploidy and poor clinical
outcome (193). Based on this association with cancer, small molecule inhibitors targeting Aurora
A or Aurora kinase family members have been developed and tested in clinical trials (200). Pan
Aurora kinase inhibitors VX680 and PHA739358 result in decreased histone H3 phosphorylation
and increased polyploidy followed by apoptosis (201). Inhibitors that work against both Aurora A
and Aurora B, including Cyc116, ZM447439 and AT9283, result in aneuploidy, disrupted cell
cycle progression, and cell death (201). Inhibitors with a high specificity for Aurora A such as
ENMD2076, MK5108, and MLN8237 (commercially known as Alisertib) had varying effects but
generally resulted in inhibition of mitotic progression usually affecting the mitotic spindle, cell
cycle inhibition, and cytotoxicity (201). Unfortunately, these Aurora A inhibitors have shown
activity against other kinases, making it difficult to discern the true effect of Aurora A inhibition
(201). For example, Alisertib, which is purported to be highly specific to Aurora A, has been
shown to concurrently inhibit Aurora B at higher concentrations (202). Additionally, response to
Aurora A inhibition appears to be dependent on p53 status (202, 203) .
Previous students from our lab have shown that loss of Aurora A leads to mitotic arrest and
incomplete mitosis resulting in cell death (204, 205). The overall effect was a decrease in the
proliferating tetraploid population and overall incidence of tetraploidy and aneuploidy which could
reduce tumor evolution and heterogeneity. This effect makes Aurora A an attractive target for
therapy in cancers prone to aneuploidy including ovarian carcinomas driven by reduced BRCA1
expression. Importantly, studies by our lab and others have shown that Aurora A and BRCA1 have
62

an inverse relationship and may work synergistically in cancer (174). Because of this, inhibition
of Aurora A may be beneficial in BRCA1 mutation carriers.
To determine if Aurora A can be a suitable target for treatment in ovarian cancers in BRCA1
mutation carriers, we sought to further elucidate the role of Aurora A in mitosis using siRNA
technologies to try and determine how Aurora A downregulation can contribute to cell death. We
used siRNA technologies to avoid potential complications due to the lack of specificity of current
small molecule inhibitors. We also used our pre-cancerous cell model in order to avoid potential
complications in interpretation of our results due to the variety of unknown genetic defects
associated with cancer to investigate the role of Aurora A in relatively normal cell cycle
progression. Here we show that inhibition of Aurora A is associated with an increase in cohesion
fatigue likely due to induced mitotic arrest. Additionally, we found that decrease of Aurora A also
leads to incomplete chromosome condensation likely due to a lack of Histone H3 phosphorylation
demonstrating a novel role of Aurora A in regulating chromosome condensation and Histone H3
phosphorylation. These data give us some insight into the efficacy of Aurora A inhibition as a
chemotherapeutic target to combat the cell non-autonomous signal stimulating cell proliferation
that may influence disease progression.

MATERIALS AND METHODS

Cell Strains and Culture Conditions. The isolation and characterization of ovarian cystadenoma
cell strains transfected with SV40LTA (ML10 cells) was previously described (59). ML10 cells
were grown in Minimal Essential Medium (MEM) supplemented with 10% Fetal Bovine Serum
63

(FBS) and 50U Penicillin, 50 µg Streptomycin per ml. Cells were incubated at 37
o
C under
atmospheric conditions containing 95% humidity and 5% carbon dioxide.

Down-regulation with siRNA. ML10 cells were transfected with siRNA sequence 5’-
AUGCCCUGUCUUACUGUCA-3’ targeting Aurora A or siGFP (as previously described) for 72
hours. Transfection was achieved using Lipofectamine 2000 according to manufacturer’s protocol.

Protein extraction procedures. For nuclear separation and extraction, tissue culture dishes were
briefly rinsed with ice cold PBS, lysed in Buffer A [0.1% Triton X, 20mM N-ethylmaleimide
(NEM), 10mM HEPES pH 7.9, 10mM potassium chloride, 10mM beta-glycerophosphate 1mM
sodium vanadate, 0.5mM phenylmethanesulfonyl fluoride (PMSF), 2ug/ml aprotinin, 1.5mM
magnesium chloride, 1ug/ml leupeptin, 1ug/ml pepstatin A, and 0.5mM dithiothreitol (DTT)], and
harvested from the dish bottom by scraping. The cell lysates were rocked for 20 minutes at 4
o
C
and centrifuged at 1150 x g for 10 min at the same temperature. The supernatant was discarded,
and the cell pellet was resuspended in Buffer C [0.42mM sodium chloride, 6.25% glycerol, 20mM
NEM, 20mM HEPES pH 7.9, 1mM sodium vanadate, 10mM beta-glycerophosphate, 2ug/ml
aprotinin, 1.5mM magnesium chloride, 1ug/ml leupeptin, 1um/ml pepstatin A, 0.5mM DTT,
0.2mM ethylenediaminetetraacetic acid (EDTA), and 0.5mM PMSF]. The resuspended pellets
were rocked for 30 minutes at 4
o
C and centrifuged at 4
o
C for 30 min at 15871 x g. The supernatants
containing the nuclear proteins were collected and stored at -80
o
C until use.
For total protein extraction the cells were lysed in Triton lysis buffer (TLB pH 8.0,
containing 25mM sodium phosphate, 150 mM sodium chloride, 1% Triton X-100, 5mM EDTA
50mM sodium fluoride) freshly supplemented with 1mM PMSF, 1mM sodium vanadate, 10ug/ml
64

aprotinin, 10ug/ml leupeptin, 5uM pepstatin A, 25mM phenylarsine oxide (PAO), rocked for 30
minutes at 4
o
C and centrifuged at 4
o
C for 30 min at 15871 x g. The supernatants containing the
total proteins were collected and stored at -80
o
C until use.

Antibodies. Antibodies against Aurora A (Cell Signaling Technology, Cat#: 4718S) were used at
a 1:1000 dilution. Antibodies against Histone H3 phosphorylated at Ser10 (Santa Cruz
Biotechnology, Cat# sc-8656-R) was used at a 1:500 dilution while antibodies specific to
phosphorylation at Thr3 (Cell Signaling Technology, Cat# 9714S), Thr11 (Cell Signaling
Technology, 9764S), and Ser28 (AbCam, Cat# ab5169) were used at a dilution of 1:200.
Antibodies against total Histone H3 (Cell Signaling Technology, 9715S) were used at a dilution
of 1:500. Antibodies against GAPDH (Santa Cruz Biotechnology, sc-25778) and KU70 (Santa
Cruz Biotechnology, Cat# sc-12729) were used at a 1:500 dilution.

Still Photography Using Confocal Microscopy. Cells were grown on chamber slides and
transfected for 72hrs. Cells were then fixed and permeabilized with 4% formaldehyde-0.02%
Triton X-PBS for 30 minutes, washed, and mounted in SlowFade Gold Reagent with DAPI
(ThermoFisher, Cat# S36938) and visualized using a PerkinElmer Spinning Disk Microscope with
63X magnification and captured

Metaphase spread. Cells were transfected for 72 hours. During the final 16 hours of transfection,
cells were treated with 0.05ug of colcemid per ml of media. Cells were then stripped and washed
with PBS. Cells were treated with 6ml of 0.075M potassium chloride for 30 minutes and then fixed
by adding 150µl of 3:1 methanol:acetic acid and pelleted at 1000rpm for 8 minutes. The
65

supernatant was carefully removed, and cells were resuspended in 5ml fixative and pelleted and
this process was repeated twice. Cells were then resuspended in 1-2 ml fixative and incubated
overnight at 4°C. Slides were briefly washed with methanol followed by distilled and deionized
water and samples were then dropped onto slides slanted at a 45° angle from a distance of at least
1.5 meters. Four to five drops of fixative were added to the slide and allowed to air dry. Samples
were then mounted in SlowFade Gold Reagent with DAPI and visualized using a PerkinElmer
Spinning Disk Microscope with 63X magnification and captured

RESULTS

Loss of Aurora A leads to an increase in cohesion fatigue.
Previous students in our lab have determined that Aurora A downregulation leads to a
decrease in tetraploidy development due to cell death and mitotic arrest at metaphase (204, 205).
To better understand the mechanism of mitotic arrest and cell death, we sought to determine the
effect of Aurora A downregulation on mitotic progression, specifically metaphase to anaphase
transition. Previous studies suggest that Aurora A downregulation leads to a mitotic arrest at
metaphase despite passing the spindle assembly checkpoint (204). Based on these observations we
decided to take a closer look at the characteristics of cells in metaphase after Aurora A
downregulation. Successful downregulation of Aurora A was achieve using siRNA and assessed
by western blot of whole cell extracts (Fig. 7A). Confocal microscopy studies were conducted to
assess any abnormalities present in metaphase, such as cohesion fatigue, when Aurora A is
downregulated. Cohesion fatigue is characterized by asynchronous movement of chromosomes
toward the spindle poles due to prolonged mitotic arrest causing constant tension that prematurely
66

causes the cohesin protein to break or relax inhibiting its ability to hold sister chromatids together.
Examples of cohesion fatigue are depicted in Fig. 7B. These data show that downregulation of
Aurora A led to an increase in cohesion fatigue (Fig. 7C). In cells treated with control siRNA, only
8 out of 44 (18.2%) observed metaphases exhibited cohesion fatigue. In contrast, Aurora A
downregulation lead to cohesion fatigue in 22 out of 60 metaphases (36.7%, p-value = 0.0496).
The importance of this finding is that it confirms a prolonged mitotic arrest at metaphase in cells
with depleted Aurora A despite contradicting factors that indicate that the cell should be able to
transition from metaphase to anaphase. However, the reasons why the cell is unable to transition
is not known.

Loss of Aurora A inhibits proper chromosome condensation
One possibility explaining the metaphase arrest is chromosome entanglement (206).
During mitosis, chromosomes condense to prevent chromosome entanglement as sister chromatids
congress at the metaphase plate and eventually separate during anaphase. Chromosome
entanglement due to insufficient chromosome condensation could potentially inhibit proper sister
chromatid segregation and thus anaphase progression (207). To determine if this is a viable
explanation, we sought to determine whether defects in chromosome condensation occurred after
Aurora A downregulation by metaphase spreads. Examples of complete and incomplete
chromosome condensation in metaphase spreads are shown in Fig. 8A where incomplete
chromosome condensation is characterized by elongated chromosome arms. Analysis of
chromosomes from these metaphase spreads showed that downregulation of Aurora A leads to an
increase in incomplete chromosome condensation (Fig. 8B). In cells treated with control siRNA,
incomplete chromosome condensation was seen in 5 out of 50 viewed metaphase spreads (10%).
67

Aurora A downregulation resulting in 3 times more incidence of incomplete chromosome
condensation (15 out of 50, 30%, p-value = 0.0228). This result is significant as it correlates with
the incidence of cohesion fatigue. These results show that downregulation of Aurora A results in
defects in chromosome condensation.

Aurora A regulates Histone H3 phosphorylation
Chromosome condensation is orchestrated by several events, one of which being histone
H3 phosphorylation. Because Aurora A is a kinase, we sought to determine if histone H3
phosphorylation could be targeted or regulated by Aurora A. Several phosphorylation sites on
histone H3 are involved in chromosome condensation: Thr3, Ser10, Thr11, and Ser28 (208).
Western blot of nuclear extracts from cells treated with siRNA against GFP (control) and Aurora
A were probed using antibodies specific for each phosphorylation modification on histone H3 (Fig.
8C). We found that, of these sites, Thr3, Thr11, Ser28 were negatively affected by Aurora A
downregulation. Conversely, decrease in Aurora levels leads to an increase in Ser10
phosphorylation. This effect is likely due to the increase of cells arrested in mitosis. These data
demonstrate a novel role of Aurora A directly or indirectly regulating chromosome condensation
through histone H3 phosphorylation.

DISCUSSION

The potential therapeutic use of Aurora A inhibitors as anti-tumorigenic agents gives cause
for researchers and clinicians to better understand the physiological effects and changes that
Aurora A inhibition causes to validate its use and predict potential unintended effects. While
68

Aurora A is upregulated in several cancers having an oncogenic effect, Aurora A is still critical
for normal cellular processes, so total obliteration of Aurora A is likely to have negative side
effects on non-target cells. Findings by a previous student determined that downregulation of
Aurora A reduced incidence of prolonged mitotic arrest, multinucleation, and proliferating
tetraploidy in our aging cell population (204). Here we found that in the younger cell population,
downregulation of Aurora A lead to an increase in cell death and cohesion fatigue, a precancerous
lesion. Thus, downregulation in young healthy cells may be detrimental to the health of the cells.
This suggests that prophylactic treatment of patients with Aurora A inhibitors may not be a viable
option for at risk patients until certain physiological changes begin to emerge, as with older patients
of in premalignant lesions in the fallopian tubes. These physiological changes can be used as
biomarkers to determine which candidates are appropriate for Aurora A inhibition treatment as a
preventative measure to prevent the emergence of genomic instability and thus prevent oncogenic
transformation.
In a previous publication, we determined that downregulation of BRCA1 in our cell model
lead to an increase in genomic instability, namely multinucleation and tetraploidy (173). Because
Aurora A is upregulated in many ovarian cancers, current research is exploring the potential
usefulness of Aurora A inhibition in treatment in ovarian cancers with BRCA1 mutation. A recent
publication has elucidated some of the regulatory interactions of concurrent Aurora A/B and
BRCA1/2 downregulation (174). They found that Aurora kinases and BRCA protein expressions
are inversely related. Similar to our current and previously published results, the authors also found
that Aurora downregulation leads to a decrease in abnormal cytokinesis, multinucleation, and
tetraploidy development while BRCA downregulation lead to defects in cytokinesis and increased
multinucleation and tetraploidy. However, the concurrent downregulation of Aurora A/B and
69

BRCA1/2 negated the effects of individual inhibition. To further elucidate the mechanism by
which Aurora A inhibition leads to decreases in multinucleation, tetraploidy, and defective
cytokinesis, our work has shown that Aurora A downregulation leads to cell death which prevents
the perpetuation of genetic instability, especially in precancerous cells. Additionally, in aging cells
with genomic instability and decreased BRCA1 expression, downregulation of Aurora A lead to a
decrease in cohesion fatigue which could promote defective mitosis. Interpreting these data, it can
be suggested that Aurora A inhibition may work best in patients with normal BRCA1 expression
or who exhibit stable karyotypes. These studies together can help define a patient population in
which Aurora A inhibition therapy is an optimal option.
Of the many roles that Aurora A plays in mitotic regulation, these data show that Aurora
A may be directly or indirectly involved in chromosome condensation. Loss of Aurora A leads to
incomplete chromosome condensation in metaphase spreads and decreases phosphorylation at
Histone H3 at Thr3, Thr11, and Ser28 but not Ser10. Phosphorylation at Ser10, Thr11 and Ser28
are strongly corelated to chromosome condensation during both mitosis and meiosis (209-211).
Interestingly, phosphorylation of Histone H3 at Thr3, which previously had been thought to be
mediated by haspin, is found to occur in prophase but is dephosphorylated during anaphase when
chromosomes are thought to be their most condensed (212). Aurora B has been found to be
responsible for Ser10 and Ser28 phosphorylation (210). It is believed that the Death‐associated
protein (DAP)‐like kinase (Dlk) or Zipper interacting protein (ZIP) kinase is responsible for Thr11
phosphorylation (211). We think it is possible that Aurora A regulates the activity of these kinases
which in turn regulate Histone H3 phosphorylation. We hypothesize that this incomplete
chromosome condensation can cause chromosome entanglement in metaphase which may in turn
cause a prolonged mitotic arrest resulting in cohesion fatigue. If cohesion fatigue impedes proper
70

mitosis, the cells may trigger a cell death response to prevent the emergence of precancerous
lesions such as tetraploidy and other forms of genomic instability.
Together these data can help optimize treatment for cancer patients by identifying patients
where Aurora A inhibition therapy would work best based on the progress of the disease or genetic
background of the patients. This type of research will help personalize therapy and thereby increase
successful treatment.

71

Figure 6: Aurora A downregulation leads to cohesion fatigue

Figure 7: Aurora A downregulation leads to cohesion fatigue. A: Western blot of total protein
extracts of cells after transfection with siRNA against GFP (siGFP) or against Aurora A (siAA)
probed with antibodies against Aurora A to confirm protein knockdown. GAPDH is used as
loading control. B: Confocal microscopy of ovarian cystadenomas in metaphase stained with
DAPI depicting normal metaphase and cohesion fatigue. C: Cystadenomas transfected with
siRNA against either GFP or Aurora A were examined by confocal microscopy and cells in
metaphase were scored on whether it depicted a normal mitosis or cohesion fatigue; the numbers
on the stacked bars represent the total number of cells for each parameter
72

Figure 7: Aurora A downregulation leads to defects in chromosome condensation
Figure 8: Aurora A downregulation leads to defects in chromosome condensation. A: Example
of metaphase spreads showing complete chromosome condensation and incomplete chromosome
condensation. B: Metaphase spreads from cystadenomas transfected with siRNA against GFP or
Aurora A were examined by confocal microscopy and scored based on the presence of
completely condensed chromosomes or incompletely condensed chromosomes; the numbers on
the stacked bars represent the total number of cells for each parameter. C: Western blot of
nuclear extracts from cystadenomas treated with siRNA against GFP or Aurora A and probed
with antibodies against Histone H3 phosphorylation at Thr3, Ser10, Thr11, and Ser28. Ku70 is
used a protein loading control and endogenous levels of Histone H3 was used reference for total
protein present.
73

CHAPTER 4: SUMMARY AND FUTURE DIRECTIONS

Understanding and controlling the emergence of aneuploidy development is essential to
better understanding disease development in ovarian carcinogenesis. In these studies, we have
shown that prolonged mitotic arrest due to defects in microtubule anchoring contributing to
cohesion fatigue mostly leads to cell death but may also result in some cells abruptly exiting this
arrest and failing cytokinesis creating tetraploid cells. Loss of BRCA1 can contribute to aneuploidy
development since it leads to increased microtubule anchoring resulting in erroneous attachments
that can cause tension on chromosomes causing the DNA to unravel and form chromosome bridges
that can interfere with cytokinesis resulting in cytokinesis failure and the development of
tetraploidy which can contribute to aneuploidy.
We have yet to elucidate why microtubule anchoring is defective in aging cell populations.
Because replicative aging is associated with decreases in BUB1, I attempted to recapitulate the
mitotic arrest by downregulating BUB1 in our younger cell population (unpublished preliminary
data). However, this was not successful in forcing a mitotic arrest. It is possible that a minimal
level of BUB1 needs to remain in order to induce a mitotic arrest, as complete loss of BUB1 would
prevent MCC formation and would essentially abolish the spindle assembly checkpoint complex.
Some sort of controlled approach to BUB1 downregulation could be successful in recapitulating
the arrest as our data suggests that there may be enough BUB1 to orchestrate MCC formation and
thus initiate assembly of the spindle assembly checkpoint complex in aging cell populations, but
not enough to phosphorylate BUB3 and recruit microtubules to the kinetochore.
My initial findings regarding BUB1 are correct, we must consider other factors that may
contribute to defects in microtubule anchoring. One possibility is that there is a defect in the
74

apparatus responsible for capturing microtubules such as the components of the kinetochore.
Defects in kinetochore formation, particularly those affecting the outer kinetochore, would have a
negative effect on microtubule anchoring. For instance, studies in Saccharomyces cerevisiae have
shown that mutants lacking components of the Ndc80 complex component of the fibrous corona
of the outer kinetochore have defects in kinetochore-microtubule attachment (213, 214).
Identifying a particular component of the analogous human structure that can prevent microtubule
anchoring without impeding MCC formation (particularly since the MCC is formed on the
kinetochore) may be key to identifying the cause of the mitotic arrest in aging cells.
One aspect of our findings that requires further study is the role of the chromosomal
passenger complex in correcting erroneous microtubule anchoring when BRCA1 levels are altered.
In our findings, decreases in BRCA1 levels led to an increase in microtubule binding that likely
resulted in microtubule abnormalities leading to the formation of chromosome bridges. However,
erroneous attachments are supposed to be corrected by the CPC (215) while our results suggest
that this does not occur. Why the chromosome passenger complex is unable to correct these
attachments when BRCA1 is downregulated needs to be investigated. One possibility is that the
chromosome passenger complex is unable to correct these erroneous attachments because
merotelic attachments, which we suspect to occur with BRCA1 downregulation, do not trigger the
spindle assembly checkpoint which would normally halt mitotic progression until the attachments
are corrected. Another possibility is that components of the chromosome passenger complex may
be regulated by BRCA1 at the transcriptional level, since it has been suggested that BRCA1
interacts with human RNA Polymerase II (216, 217) (though this has been challenged by a study
that proposed that the predominant complex that BRCA1 interacts with is the SWI/SNF complex,
which is involved with chromatin remodeling, though this could still affect transcription by
75

altering access to DNA near nucleosomes (218, 219)). Conversely, BRCA1 has been found to
interact with NELF-B (also known as cofactor of BRCA1, COBRA1) which is a subunit of the
negative elongation factor (NELF) which inhibits transcription by RNA polymerase II (220).
Given the effect of BRCA1 downregulation on microtubule anchoring, future research
should focus on ways to correct such anchoring abnormalities to prevent cytokinesis failure. One
approach would be to enhance or optimize the ability of the chromosome passenger complex to
correct erroneous attachments. Upregulation of chromosome passenger complex components,
particularly Aurora B, which is responsible for correcting syntelic attachments that are potential
precursors of merotelic attachments, might prevent mitotic progression in the presence of
erroneous microtubule attachment by increasing the availability of chromosome passenger
complexes to search for and correct erroneous microtubule anchorings. Another approach could
be to prevent premature release from the spindle assembly checkpoint in the presence of merotelic
attachment by upregulating MAD2 in the closed conformation to better sequester CDC20,
preventing activation of the APC, or by upregulating or stabilizing cyclin B1 to make cyclin B1
degradation more difficult hindering transition from metaphase to anaphase. In fact, cyclin
dependent kinases have recently been studied as potential targets in ovarian cancer treatment (221,
222). Another tactic could be to destabilize microtubule anchoring by downregulating EB1. This
end-binding protein accumulates at the kinetochore upon microtubule anchoring, stabilizing
attachments. Decreased of EB1 might make these attachments less stable, making them more
susceptible to dynamic microtubule interactions and thus correction.
Because Aurora A is upregulated in several cancers, attempts to inhibit disease
development through inhibition of Aurora A have been made. Interestingly, published data as well
as our own unpublished observations showed that Aurora A and BRCA1 are inversely related (174,
76

204, 205), making Aurora A an interesting target in hereditary cases of ovarian cancer. However,
since Aurora A is necessary for normal cellular processes, it is imperative to elucidate the effect
of Aurora A downregulation on mitotic progression specifically. My data show that loss of Aurora
A leads to a mitotic arrest that results in cohesion fatigue that may promote cell death. Additionally,
I have demonstrated a novel role of Aurora A in regulating chromosome condensation. However,
whether this effect is direct or indirect is unknown.
The role of Aurora A in chromosome condensation should be investigated further. My data
show that Aurora A plays a role in regulating the phosphorylation of several key sites on Histone
H3 that are known to be involved in chromosome condensation. However, it is not known if Aurora
A phosphorylates these sites directly or interacts with or activates other kinases that are responsible
for phosphorylating Histone H3. Phosphorylation at Ser10 and Ser28 of this histone is carried out
by Aurora B (210). Haspin is responsible for phosphorylation of Histone H3 at Thr3 and also
recruits Aurora B to the centromeres though loss of haspin has no effect on Ser10 phosphorylation
and thus does not play a role is this interaction (212). Lastly, Histone H3 Thr11 phosphorylation
is attributed to Dlk/ZIP kinase. Given that each of these sites are phosphorylated by different
kinases while, in most cases, also being influenced by downregulation of Aurora A, it seems that
either these kinases are under the regulation of Aurora A or that Aurora A stabilizes their
phosphorylated products.
I proposed that loss of Aurora A may inhibit mitotic progression by causing chromosome
entanglement at metaphase, preventing proper chromosome segregation despite spindle assembly
checkpoint complex inactivation. Unfortunately, there currently are no suitable techniques for
studying how defects in chromosome condensation may contributes to chromosome entanglement.
Techniques for investigating chromosome entanglement need to be developed. Such a technique
77

may involve antibody coated magnetic bead capture of centrosomes that can then be manipulated
by magnets or lasers to pull anchored chromosomes in metaphase apart in the presence of
paclitaxel or MG132, a proteasome inhibitor, which would arrest the cell in metaphase. In normal
mitotic cells, chromosomes should readily separate. If loss of Aurora A leads to chromosome
entanglement, chromosomes should not separate easily. Tension generated by the pulling force of
the magnets could also be measured to determine which cells exhibit entanglement. Interestingly,
other labs have shown that, in Tetrahymena thermophila and S. Pombe, mutation of Histone H3
Ser10, which would prevent its phosphorylation and thus inhibit complete chromosome
condensation, leads to abnormal chromosome segregation and extensive chromosome loss during
mitosis and meiosis, possibly illustrating how chromosome entanglement could lead to defects in
chromosome separation (223-225).
In conclusion, these studies have furthered our understanding of how defects in mitotic
regulation can lead to aneuploidy and how loss of BRCA1 can promote aneuploidy development,
providing insights into a role for BRCA1 in carcinogenesis that is independent of its role in DNA
damage repair. I have also described a novel role of Aurora A in chromosome condensation,
providing an opportunity to better understand the impact of chromosome condensation on mitotic
progression. These data also give us a better understanding of how Aurora A may be able to
counteract the effect of the cell autonomous mechanisms contributing cancer predisposition in
BRCA1 mutation carriers as illustrated in Fig. 9.

78

Figure 8: Working model for prevention of aneuploidy development by interruption of the interplay between cell-nonautonomous
and cell-autonomous mechanisms of cancer predisposition in BRCA1 mutation carriers by Aurora A inhibition
79

Figure 9: Working model for prevention of aneuploidy development by interruption of the
interplay between cell-nonautonomous and cell-autonomous mechanisms of cancer predisposition
in BRCA1 mutation carriers by Aurora A inhibition. As previously described, the menstrual cycle,
under the influence of ovarian granulosa cells and cells from the anterior pituitary, control the
proliferation of tissues that have an elevated cancer risk in BRCA1 mutation carriers (170). These
cell-nonautonomous signals are amplified in such carriers resulting in accelerated replicative
aging. P53 alterations, which are frequent in these tissues in BRCA1 mutation carriers, eventually
lead to mitotic arrest in aging cells due to defective microtubule anchoring. Cohesion fatigue
resulting in cell death is the most likely outcome. However, the presence of a germline BRCA1
mutation not only intensifies accelerated replicative aging, but also leads to recovery from the
mitotic arrest due to uncontrolled kinetochore attachments resulting in the formation of
chromosome bridges significantly increasing the rate of cytokinesis failure, leading to polyploidy
followed by aneuploidy and malignant transformation. Loss of Aurora A negates this effect by
inducing a mitotic arrest downstream of the SAC and inducing cell death in cells that would have
escaped the physiological arrest, thus preventing the development of aneuploidy in cells of
elevated risk.
80

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Abstract (if available)

Abstract An in vitro cell model of ovarian cancer development was previously developed and characterized in our laboratory. Previous students have used this model to show that the cells reach a mitotic arrest at the spindle assembly checkpoint as they proliferate and age in culture. Here, I show that this mitotic arrest is due to defects in microtubule anchoring associated with decreases in BUB1, the master regulator of the mitotic checkpoint complex formation. The prolonged arrest leads to cohesion fatigue that primarily resolves in cell death, though a small portion of cells undergo mitotic slippage where cellular structures at the site of cleavage furrow ingression interfere with abscission leading to cytokinesis failure and tetraploidy. Our laboratory previously showed that downregulation of BRCA1 in aging cell populations leads to cells overcoming the mitotic arrest, but only at the expense of cytokinesis failure, generating binucleated tetraploid cells. I report here that decreases in BRCA1 to levels similar to those present in human BRCA1 mutation carriers lead to an increase in microtubule anchoring, which may lead to erroneous attachment to the kinetochore resulting in severe intra-chromosomal bridging. Severe bridging results in cytokinesis failure contributing to tetraploidy and subsequently aneuploidy development, which may significantly contribute to cancer predisposition in BRCA1 mutation carriers. I also investigated Aurora A, a serine/threonine kinase that is upregulated in many cancers whose protein levels are inversely related to that of BRCA1, for its role in mitotic regulation. Previous students in our laboratory have shown that loss of Aurora A leads to mitotic arrest followed by cell death leading to a decrease in proliferating tetraploid cell populations. Here, I show that loss of Aurora A leads to an increase in cohesion fatigue and defects in chromosome condensation due to loss of phosphorylation on sites of Histone H3 that are important for chromosome condensation. I hypothesize that this defect in chromosome condensation leads to chromosome entanglement that would prevent chromosome segregation leading to cohesion fatigue resulting in cell death.

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Asset Metadata

Creator Austria, Theresa Mendoza (author)

Core Title Understanding and controlling mitotic errors leading to aneuploidy in early ovarian cancer development

School Keck School of Medicine

Degree Doctor of Philosophy

Degree Program Genetic, Molecular and Cellular Biology

Publication Date 03/01/2019

Defense Date 08/22/2018

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag aneuploidy,Aurora A,BRCA1,cytokinesis failure,OAI-PMH Harvest,ovarian cancer,tetraploidy

Format application/pdf (imt)

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Dubeau, Louis (committee chair), Stallcup, Michael (committee chair), Rice, Judd (committee member), Zandi, Ebrahim (committee member)

Creator Email taustria@sbcglobal.net,taustria@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c89-128870

Unique identifier UC11675760

Identifier etd-AustriaThe-7122.pdf (filename),usctheses-c89-128870 (legacy record id)

Legacy Identifier etd-AustriaThe-7122.pdf

Dmrecord 128870

Document Type Dissertation

Format application/pdf (imt)

Rights Austria, Theresa Mendoza

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the a...

Repository Name University of Southern California Digital Library

Repository Location USC Digital Library, University of Southern California, University Park Campus MC 2810, 3434 South Grand Avenue, 2nd Floor, Los Angeles, California 90089-2810, USA