Fallopian tube initiation of high grade serous ovarian cancer and ovarian metastasis: Mechanisms and therapeutic implications

Journal Pre-proof Fallopian tube initiation of high grade serous ovarian cancer and ovarian metastasis: Mechanisms and therapeutic implications Tova M. Bergsten, Joanna E. Burdette, Matthew Dean PII:

S0304-3835(20)30080-X

DOI:

https://doi.org/10.1016/j.canlet.2020.02.017

Reference:

CAN 114699

To appear in:

Cancer Letters

Received Date: 4 December 2019 Revised Date:

31 January 2020

Accepted Date: 13 February 2020

Please cite this article as: T.M. Bergsten, J.E. Burdette, M. Dean, Fallopian tube initiation of high grade serous ovarian cancer and ovarian metastasis: Mechanisms and therapeutic implications, Cancer Letters, https://doi.org/10.1016/j.canlet.2020.02.017. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Elsevier B.V. All rights reserved.

Ovarian cancer is the most lethal gynecologic malignancy and the fifth leading cause of cancer-related death in women. Although outcomes have improved in recent years, there remains an unmet clinical need to understand the early pathogenesis of ovarian cancer in order to identify new diagnostic approaches and agents of chemoprevention and chemotherapy. While high grade serous ovarian cancer (HGSOC), the most abundant histotype, was initially thought to arise from the ovarian surface epithelium, there is an increasing body of evidence suggesting that HGSOC originates in the fallopian tube. With this new understanding of cell of origin, understanding of disease development requires analysis with a novel perspective. Currently, factors that drive the initiation and migration of dysplastic tubal epithelial cells from the fallopian tube to the ovary are not yet fully defined. These factors include common mutations to fallopian tube epithelial cells, as well as factors originating from both the fallopian tube and ovary which are capable of inducing transformation and dissemination in said cells. Here, we review these changes, their causative agents, and various potential means of intervention.

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Fallopian tube initiation of high grade serous ovarian cancer and ovarian metastasis: Mechanisms and therapeutic implications Tova M. Bergsten1,2, Joanna E. Burdette2, and Matthew Dean3 1

Medical Scientist Training Program, University of Illinois at Chicago College of Medicine, Chicago, IL; 2Department of Pharmaceutical Sciences, Center for Biomolecular Science, University of Illinois at Chicago, Chicago, IL; 3Department of Animal Science, University of Illinois at Urbana-Champaign, Urbana, IL Short Title: Interaction of the ovary and fallopian tube in serous cancer Pages Abstract Figures Tables Words #

32 178 2 1 4914

Correspondence to:

Matthew Dean Department of Animal Science University of Illinois at Urbana-Champaign 1207W Gregory Drive Urbana, IL 61801 217.300.7600 [email protected] Disclosures: The authors have no conflicts to disclose.

1 2 3 4 5 6 7 8 9 10 11 12 13 14

Author Contributions: TB collated literature, assembled figures, and drafted the manuscript. JB and MD provided oversight, funding support, and edited the manuscript. Grant Support: This work is supported by grant CA240301 and startup funds provided by the University of Illinois Urbana-Champaign. Abbreviations: High grade serous ovarian cancer – HGSOC; secretory cell outgrowths – SCOUTs; serous tubal intraepithelial carcinomas – STICs; Transforming Growth Factor β TGFβ; activation-induced cytidine deaminase – AID; insulin-like growth factor 2 – IGF2; interleukin 8 – IL-8; intrauterine device – IUD; serum cancer antigen 125 – CA-125; epithelial to mesenchymal transition – EMT; The Cancer Genome Atlas – TCGA; paired box transcription factor 8 – PAX8; histone deacetylase inhibitors – HDACis; stromal cell-derived factor 1 – SDF1; progesterone – P4

-2-

ABSTRACT

1 2 3

Ovarian cancer is the most lethal gynecologic malignancy and the fifth leading cause

4

of cancer-related death in women. Although outcomes have improved in recent years, there

5

remains an unmet clinical need to understand the early pathogenesis of ovarian cancer in

6

order to identify new diagnostic approaches and agents of chemoprevention and

7

chemotherapy. While high grade serous ovarian cancer (HGSOC), the most abundant

8

histotype, was initially thought to arise from the ovarian surface epithelium, there is an

9

increasing body of evidence suggesting that HGSOC originates in the fallopian tube. With this

10

new understanding of cell of origin, understanding of disease development requires analysis

11

with a novel perspective. Currently, factors that drive the initiation and migration of dysplastic

12

tubal epithelial cells from the fallopian tube to the ovary are not yet fully defined. These

13

factors include common mutations to fallopian tube epithelial cells, as well as factors

14

originating from both the fallopian tube and ovary which are capable of inducing

15

transformation and dissemination in said cells. Here, we review these changes, their

16

causative agents, and various potential means of intervention.

17

Key Words: carcinogenesis, chemoprevention, chemotherapy, oviduct, STIC, TP53

18 19

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1 2 3

1. INTRODUCTION Ovarian cancer is the fifth leading cause of cancer-related death in women. While

4

outcomes have improved in recent years, the current five-year survival rate remains a dismal

5

47% [1]. This is largely attributed to the late stage of presentation, as the majority of ovarian

6

cancer patients are diagnosed with metastatic disease, at which point the five-year survival

7

rate can be as low as 17% [2]. As such, there remains an urgent need to clarify the early

8

pathogenesis of ovarian cancers in hopes of identifying new diagnostic techniques, as well as

9

effective means of prevention and treatment.

10

Clinically, ovarian cancers are segregated into five predominant histotypes, the most

11

common being high-grade serous ovarian cancer (HGSOC). HGSOC carries a particularly

12

poor prognosis and is responsible for 70-80% of ovarian cancer deaths [3]. Though HGSOC

13

was initially thought to arise from the ovarian surface epithelium, there is now clear evidence

14

that HGSOC can also originate in the fallopian tube epithelium [4-12]. This is significant, as

15

the cell of origin alters screening modalities, imaging practices, surgical intervention, and

16

target-based drug discovery. Ultimately, identifying the cells of origin will better enable

17

detection of pre-invasive disease and intervention before these cells disseminate and invade

18

the ovary.

19

Recent evidence supports that many HGSOCs originate in the epithelial cells at the

20

distal tips of the fimbriae of the fallopian tube, known as the oviduct in non-human species.

21

For instance, approximately 40-60% of HGSOC patients harbor additional lesions in the

22

fallopian tube [5-7]. As these lesions typically share identical TP53 mutations with their

23

HGSOC counterparts, they likely represent the clonal origin of HGSOC [8, 9]. Additionally,

24

salpingectomy is associated with a 35% reduction in ovarian cancer risk while tubal ligation

25

reduces risk by 13% [10, 11]. In a study comparing HGSOC specimens to normal epithelial

26

tissues from several other organs with cDNA microarrays, HGSOC mRNA expression -4-

1

patterns closely resembled that of normal fallopian tube epithelial cells yet differed from other

2

sites including the ovary [12]. This was paralleled by additional studies using RNA

3

sequencing, further confirming the high degree of similarity between HGSOC and the

4

fallopian tube epithelium [9].

5

Hence, while it is now understood that HGSOC can originate from fallopian tube

6

epithelial cells, how these cells initially transform and disseminate to colonize the ovary

7

remains unclear. Understanding the local cues that direct these processes may present a

8

new opportunity for intervention. In this review, we will survey the known local cues that

9

mediate both physiologic and pathologic crosstalk between the ovary and fallopian tube and

10

discuss potential means of intervention.

11

The ovary releases follicles and secretes associated hormones into and onto the

12

fallopian tube in the form of follicular fluid, which contains a variety of factors now believed to

13

mobilize dysplastic fallopian tube epithelial cells, potentially recruiting them to the ovary via

14

chemotactic gradient. Here, we describe the initial genetic insults common to ovarian cancer

15

patients, as well as the contents of fallopian tube and ovarian secretions and follicular fluid

16

that are reported to direct these abnormal cells to the ovary where they establish overt

17

tumors (Figure 1).

18

2. INITIATION OF CANCER IN THE FALLOPIAN TUBE EPITHELIUM

19

One of the earliest potential HGSOC precursor lesions, dubbed secretory cell

20

outgrowths (SCOUTs), consist of at least 30 secretory fallopian tube epithelial cells and are

21

located all throughout the fallopian tube [13]. SCOUTs are almost entirely devoid of PAX2, a

22

transcription factor normally expressed in fallopian tube epithelial cells [13-16]. Interestingly,

23

while SCOUTs are more frequently found in the fallopian tubes of patients with HGSOC, their

24

exact relationship to HGSOC has yet to be determined [17]. The Cancer Genome Atlas and a

25

follow up analysis have reported that 96-100% of HGSOC carry TP53 mutations [18]. As -5-

1

TP53 is a tumor suppressor, alteration to the gene via mutation or deletion could enable

2

unchecked cell growth, reduced DNA repair, and eventual development of cancer. The most

3

accepted preneoplastic lesion identified in relation to HGSOC is the p53 signature, consisting

4

of benign secretory cells exhibiting robust p53 staining (or a total lack of staining), positive

5

ϒH2AX staining, and lack of Ki-67 staining [19]. Serous tubal intraepithelial carcinomas or

6

STICs, share the same characteristics of DNA damage and p53 mutations as p53 signatures,

7

but are composed of malignant cells featuring a high mitotic index and disorganized growth

8

[19]. The prevailing theory is that p53 signatures progress to STICs, which are then believed

9

to ultimately develop into HGSOC [8, 19-21]. Several chemical signals have been suggested

10

to induce transformation in fallopian tube epithelial cells and subsequently play a role in

11

HGSOC initiation.

12

2.1 FOLLICULAR FLUID

13

While the association of decreased ovulation with decreased risk of developing

14

ovarian cancer is well-established epidemiologically, it remains unexplained biologically.

15

During ovulation, ovarian follicles release fluid which washes the surrounding tissues

16

including the fallopian tube. Secreted factors found in follicular fluid may be involved in the

17

initiation or development of ovarian cancer. While some components of follicular fluid and

18

their effects on neighboring tissues are discussed below, other components such as follicle

19

stimulating hormone, anti-Mullerian hormone, luteinizing hormone, and androgens, are

20

covered in depth by Emori and Drapkin [22].

21

Ovulation may instigate ovarian cancer development due to the release of follicular

22

fluid on fallopian tube epithelia. When fallopian tube epithelial cells were treated with follicular

23

fluid, they demonstrated a 1.7-fold increase in phospho-ϒH2AX, a known DNA double-

24

stranded break response [23]. Additionally, compared to negative controls, 5% of the follicular

-6-

1

fluid treated cells stained positive for TP53, suggesting that follicular fluid may induce TP53

2

stabilization [23]. This is noteworthy due to the common presentation of TP53 mutations in

3

ovarian cancer precursor lesions. Interestingly, human fallopian tube epithelial cells express

4

activation-induced cytidine deaminase (AID), which creates DNA mutations by converting a

5

C:G base pair to a T:A base pair [24]. When treated with follicular fluid, fallopian tube

6

epithelial cells demonstrated a significant induction of AID, an increase in double-strand DNA

7

breaks, and a reduction in DNA methylation [24]. Similarly, when fallopian tube epithelial cells

8

were exposed to follicular fluid, an increase in expression of APOBEC cytidine deaminases,

9

which also cause DNA mutations, was reported [25]. Transient overexpression of a particular

10

APOBEC cytidine deaminase, APOBEC3a, was able to induce double-strand DNA breaks in

11

fallopian tube epithelial cells [25]. Collectively, these DNA damaging responses to follicular

12

fluid support its potential role as a mutagen.

13

Reactive oxygen species, either contained within or produced as a response to

14

follicular fluid, have also been suggested as instigating factors in the transformation of

15

fallopian tube epithelial cells. In a model of ovulation, where tissue was collected 12 hours

16

post injection with human chorionic gonadotropin, murine oviductal epithelial cells

17

demonstrated increased levels of phospho-ϒH2AX, suggesting an increase in DNA damage

18

[26]. Pro-inflammatory macrophages, which are known to produce reactive oxygen species

19

were detected after ovulation [26]. Furthermore, follicular fluid treatment of fallopian tube

20

secretory epithelial cells induced an increase in intracellular reactive oxygen species [27].

21

Treatment of cells harboring a p53 mutation with excess reactive oxygen species led to an

22

increase in miR-182 expression which allowed cells to bypass senescence [27]. Together

23

these data suggest that the release of reactive oxygen species-containing follicular fluid

-7-

1

during ovulation may directly induce DNA damage in tubal epithelial cells or recruit pro-

2

inflammatory immune cells.

3

In addition to studying the effects of follicular fluid as a whole, its specific components

4

have also been investigated. Transferrin, an iron transport protein, has been identified as a

5

component in follicular fluid that may induce DNA damage. Treatment of fallopian tube

6

epithelial cells with transferrin increased expression of phospho-ϒH2AX and formation of

7

double-strand breaks while knockdown of the transferrin receptor or treatment with small

8

interfering RNA abrogated this effect [28]. Ferryl hemoglobin, found in ovulatory follicular and

9

peritoneal fluids, rescued p53-deficient oviduct epithelial cells from lethal reactive oxygen

10

species by consuming these species extracellularly and reducing NADPH oxidase-mediated

11

cell death [29]. However, while the cells exposed to high levels of reactive oxygen species

12

were able to evade apoptosis due to the protection by ferryl hemoglobin, they still harbored

13

double-stranded DNA breaks suggesting that their rescue could allow these damaged cells to

14

transform [29]. Similarly, fallopian tube epithelial cells treated with non-transferrin bound iron

15

displayed increased cell number, migration, and DNA damage relative to untreated cells [30].

16

Iron treated cells also demonstrated increased expression of oncogenic markers such as β-

17

catenin, Myc, and ecotropic virus integration site 1 [30].

18

Insulin-like growth factor 2 (IGF2) is a peptide hormone found in follicular fluid [31].

19

When fallopian tube epithelial cells were treated with follicular fluid, anchorage independent

20

growth increased, an effect abrogated by inhibition of the IGF receptor [31]. Interestingly, a

21

clinical study noted that IGF2 expression was higher in ovarian tumor tissues than those of

22

healthy ovaries [32]. Higher expression of IGF2 was also associated with decreased overall

23

survival in patients [32]. A separate study observing the effect of follicular fluid on fallopian tube

24

epithelial cells noted rapid upregulation of the inflammatory cytokine interleukin-8 (IL-8) [23]. Beyond

-8-

1

its canonical roles in inflammation, IL-8 has also been implicated in a variety of additional cell

2

processes in ovarian tissue, namely increased proliferation. Furthermore, Transforming Growth

3

Factor β (TGFβ) has also been identified as a component of follicular fluid for which fallopian

4

tube epithelial cells express receptors. One study observing the effect of TGFβ on oviductal

5

epithelium demonstrated a reduction in PAX2 expression, which could potentiate precursor

6

lesions through loss of tumor suppressive action [33]. These data suggest that while some

7

components of follicular fluid could contribute to the initiation of ovarian cancer, others could

8

play a role in tumor maintenance by promoting proliferation.

9

As previously mentioned, decreased ovulation is associated with decreased risk of

10

developing ovarian cancer. In concordance, epidemiologic data also suggests that the use of

11

oral contraception reduces ovarian cancer risk since its components, progestin with or

12

without estrogen, block ovulation [34]. Intrauterine devices (IUDs), another popular

13

mechanism of contraception, prevent pregnancy through both the inflammatory response to a

14

foreign body, which is toxic to sperm and ova, as well as local effects specific to the type of

15

IUD [35]. For example, levonorgestrel IUDs release progestins which can prevent ovulation

16

and thicken cervical mucus to prevent fertilization and implantation [35]. Interestingly, a

17

retrospective review described an association between IUD use and decreased risk of

18

developing ovarian cancer [36]. However, as this study was retrospective, the specific type of

19

IUD was unavailable for subset analysis so the resulting association warrants further

20

investigation in order to frame its relation to ovulation.

21

2.2 ESTROGEN

22

The local role of estrogen on the fallopian tube primarily involves facilitating the

23

movement and maturation of eggs, sperm, and embryo rolling [37]. However, exposure to

24

estrogens, including the natural hormone estradiol, is considered a risk factor for

-9-

1

development of ovarian cancer [38-40]. Estrogen induced changes include genetic mutations,

2

altered gene expression, as well as downstream effects of estrogen-induced oxidants like

3

DNA strand breaks and adduct formation [41, 42]. Interestingly, while treatment of murine

4

ovarian epithelial cells with estradiol did not result in cellular proliferation and migration, RNA

5

sequencing of these cells revealed increased expression of proliferation and anti-apoptosis

6

transcripts [43]. Furthermore, early lesions in the fallopian tube composed of PAX2 deficient

7

cells have been shown to have increased estrogen receptor [44]. Clinically, women taking

8

estrogen only hormone replacement therapy have increased risk of developing ovarian

9

cancer compared with those who have never taken estrogen only therapy or estrogen-

10

progesterone combination therapy [38, 39]. Together, this data suggests that estrogens may

11

be involved in the development of ovarian cancer.

12

Hormonal therapy has shown efficacy in certain cancers. In the treatment of ovarian

13

cancer, various clinical studies analyzing both antiestrogens and inhibitors of aromatase,

14

which converts testosterone to estradiol, have demonstrated modest efficacy [45]. This is

15

supported by a separate analysis of 53 different trials of endocrine therapies which

16

demonstrated a clinical benefit rate of 41%, with an increased effect in estrogen receptor

17

positive tumors [46]. Additionally, several clinical trials have shown that tamoxifen provides a

18

response rate of 15% in platinum-resistant ovarian cancer which has led to its use as a

19

second-line treatment since its toxicity profile is more favorable than that of other

20

chemotherapy agents [47]. However, studies of endocrine therapies require more robust

21

analysis pertaining to subtype sensitivity and optimal treatment settings.

22

2.3 PROGESTERONE

23

Progesterone, like estrogen, is a steroid hormone component of follicular fluid which is

24

necessary for proper follicle development and ovulation. Progesterone is believed to mainly

25

have a protective effect against ovarian cancer. In studies of patients with ovarian cancer, -10-

1

increased progesterone receptor expression was associated with improved overall and

2

disease-free survival [48, 49]. This is further supported by the observation that women taking

3

combined estrogen-progestin hormone replacement therapy or oral contraceptives have

4

decreased relative risk of ovarian cancer when compared with non-users [50-52].

5

Additionally, pregnancy is associated with decreased risk of ovarian cancer, an observation

6

likely due to higher levels of progesterone during pregnancy [53].

7

3. FACTORS OF FALLOPIAN TUBE ORIGIN AND THEIR ROLE IN DISEASE

8

The change in paradigm regarding cell of origin creates an opportunity to study

9

disease onset and progression from an angle that had previously been neglected. For

10

instance, as recent research has found, there are several factors that originate in the fallopian

11

tube that both serve as a marker of origin for HGSOC and to influence disease progression.

12

3.1 CA-125

13

Serum cancer antigen 125 (CA-125), a glycoprotein also known as MUC16, is

14

expressed in epithelial cells in normal tissues like the genitourinary tract and overexpressed

15

in some epithelial cancers [54]. MUC16 expression in epithelial ovarian cancers aligns with

16

that of fallopian tube epithelium, suggesting the latter as the cell of origin [55]. CA-125 is

17

currently utilized as a surrogate biomarker in clinical practice to gauge response to treatment

18

and detect recurrence [56]. However, CA-125 is not used presently for ovarian cancer

19

screening.

20

In addition to its use as a surrogate biomarker, CA-125/MUC16 appear to contribute to

21

disease development. For example, knockdown of MUC16 in ovarian cancer cells resulted in

22

increased synapses between these and natural killer cells, and increased lysis of ovarian

23

cancer cells [57]. In vivo, mice with ovarian tumors featuring MUC16-knockdown displayed

24

increased survival compared to controls [58]. These data suggest that CA-125/MUC16 are

25

capable of suppressing the innate anti-tumor immune system response. Another suggested -11-

1

role for CA-125 is in metastasis, since stable expression of MUC16 increased motility and

2

invasiveness [59]. CA-125 was also associated with decreased expression of E-cadherin and

3

increased expression of N-cadherin and vimentin suggesting its effects on metastasis and

4

epithelial to mesenchymal transition (EMT) [59]. Additionally, down-regulation of MUC16 led

5

to increased susceptibility of ovarian cancer cells to cisplatin [60]. While similar effects were

6

not noticed in response to paclitaxel, this data supports the role of MUC16 in genotoxic

7

chemotherapy resistance [60].

8

Several anti-CA-125/MUC16 therapies have been investigated. One such potential

9

treatment aimed to target MUC16-mesothelin interactions via a fusion of human antibody Fc

10

region to the binding domain of mesothelin, but it remains to be examined in a clinical setting

11

[61]. In patients with recurrent ovarian cancer, co-treatment of chemotherapy and

12

oregovomab, a murine monoclonal antibody directed against CA-125, resulted in immune

13

responses to CA-125 and improved time to progression and survival [62]. Another strategy

14

utilizing DMUC5754A, composed of an anti-MUC16 antibody conjugated to a microtubule-

15

disrupting agent, demonstrated anti-tumor activity in patients with MUC16 expressing tumors

16

with an acceptable safety profile in a Phase I clinical trial [63].

17

3.2 PROLACTIN

18

Prolactin, most commonly referenced as a neuroendocrine hormone secreted from the

19

anterior pituitary, is also produced in many peripheral tissues including the brain, mammary

20

glands, placenta, amnion, decidua, and uterus [64]. Similarly, prolactin receptors are found

21

throughout the body [64]. Interestingly, both human ovaries and fallopian tube epithelia

22

produce prolactin and express prolactin receptors indicating that prolactin may act as a

23

hormone and auto- or paracrine regulatory factor [65, 66].

24

A clinical study noted that compared with pancreatic, lung, and breast cancers,

25

prolactin levels were highest in patients with endometrial and ovarian cancers [67]. In a study -12-

1

of 230 patients with ovarian cancer, 120 of which had serous histology, increased prolactin

2

was associated with an increased risk of ovarian cancer in the subgroup of patients with a

3

BMI > 25kg/m2 [68]. Recently, a prolactin receptor targeted imaging platform has

4

demonstrated more specific and sensitive detection of ovarian cancer when compared with

5

currently utilized contrast agents like GdDTPA [69].

6

The clinical data suggesting prolactin could be involved in ovarian cancer aligns with in

7

vitro data. In a murine spontaneous model of fallopian tube-derived ovarian cancer, a

8

prolactin-like gene (Prl2c2) was the most abundant transcript and silencing it reduced tumor

9

burden [70]. In the ovarian cancer cell line OV-90, prolactin receptor antagonists decreased

10

cell number and migration [71]. In both the transformed human fallopian tube epithelium cell

11

line FT33-TAg-Myc and the ovarian cancer cell line OVCAR8, treatment with prolactin

12

induced proliferation through the receptor [66]. When mice were injected with OVCAR3 cells

13

that had prolactin receptors deleted, no tumor growth was noted [66]. Moreover, incubating

14

OVCAR3 cells with prolactin prior to treatment with cisplatin protected the cells from drug-

15

induced apoptosis [67]. Collectively, these data indicate that since prolactin is normally

16

secreted from the fallopian tube epithelia, it likely acts in an autocrine manner to induce

17

clonal proliferation of tumorigenic cells and potentiate chemoresistance.

18

For this reason, prolactin and prolactin receptor antagonists are attractive treatment

19

modalities. Of note, when orthotopic models of human ovarian cancer were treated with the

20

prolactin antagonist G129R, the result was an inhibition of tumor growth and increase in

21

autophagy [72]. However, little research has been conducted regarding the prolactin signaling

22

pathway and its potential for therapeutic use in ovarian cancer and much remains to be

23

concluded.

24

3.3 PAX8

-13-

1

While paired box transcription factor 8 (PAX8) is normally expressed in the fallopian

2

tube epithelium, it is not usually expressed in the ovarian surface epithelium [73]. However,

3

PAX8 is almost universally expressed in HGSOC tumors [73]. These data serve as support

4

for PAX8 as a marker of cell origin, demonstrating that HGSOC can stem from fallopian tube

5

epithelial cells.

6

In addition, PAX8 also plays a role in disease development. For example, knockdown

7

of PAX8 expression in a human ovarian cancer cell line resulted in reduction of proliferation,

8

migration, and invasion, as well as tumorigenesis in an in vivo model [74]. Similarly,

9

knockdown of PAX8 through shRNA was able to reduce viability and induce apoptosis of a

10

majority of tested ovarian cancer cell lines [75]. Another study observing the effect of PAX8

11

expression in ovarian surface epithelial cells demonstrated increased proliferation and

12

migration, as well as upregulation of various factors associated with EMT [76]. Interestingly,

13

while knockdown of PAX8 in several human HGSOC cell lines decreased proliferation and

14

increased apoptosis, knockdown in oviductal epithelium had little to no effect on proliferation,

15

migration, or apoptosis [76]. This suggests an opportunity to target PAX8 pharmacologically

16

in order to reduce the viability of transformed cells without affecting normal tissues.

17

While PAX8 expression may not be a predictive biomarker as it does not associate

18

with tumor stage or disease outcome, its high prevalence in HGSOC cases makes its

19

inhibition a suitable candidate for therapeutic intervention. One such possible intervention is

20

thiostrepton, an antibiotic that inhibits translation by binding the prokaryotic ribosome, which

21

was able to reduce PAX8 and inhibit tumor progression in vivo [77]. Another investigation of

22

anti-PAX8 therapy centers around class I histone deacetylase inhibitors (HDACis).

23

Panobinostat (a pan-HDACi) and romidespin (a class I HDACi) were able to eradicate PAX8

24

protein and PAX8-positive tumor cells, as well as inhibit migration through disruption of PAX8

25

transcription and downstream regulation [78]. Further in vivo analysis showed that treatment -14-

1

with HDACis resulted in decreased tumor burden and metastatic dissemination while co-

2

treatment with HDACis and platinum chemotherapy indicated less tumor progression than

3

single therapy treatment [78]. Taken together, these data suggest PAX8 may be both a

4

marker of cell of origin and a worthwhile therapeutic target for HGSOC.

5

4. DISSEMINATION OF FALLOPIAN TUBE CELLS TO THE OVARY

6

Fallopian tube dysplastic cells likely exhibit organotropism for the ovary since the

7

majority of HGSOC patients are diagnosed with large ovarian masses. To this point, tail vein

8

injection of four separate human ovarian cancer cell lines led to a total of 66% of mice

9

developing ovarian metastases while injection of four non-ovarian cancer cell lines led to no

10

ovarian metastases [79]. This suggests that ovarian cancer cells have a particular proclivity

11

for spread to the ovary. Since a murine model showed superovulation increased the

12

colonization of the ovary following xenograft of ovarian tumor initiating cells, this

13

predisposition correlates with ovulation [80]. Additionally, when tumorigenic fallopian tube

14

epithelial cells were injected in murine bursa, all mice developed ovarian tumors and were

15

sacrificed due to tumor burden by day 90, while no mice receiving intraperitoneal injections

16

developed tumors for the duration of the experiment [81]. In transgenic mouse model which

17

spontaneously develops ovarian cancer, removal of the ovaries reduced peritoneal

18

metastases, suggesting that ovaries are required for such progression [82]. While these data

19

support the tendency for fallopian tube cells to migrate to the ovary preferentially, which

20

signaling molecules contribute to this dissemination are yet unclear. However, several

21

potential factors have been explored and are discussed in the following sections.

22

4.1 WNT Signaling

23

Recent evidence appears to implicate WNT signaling in the recruitment of abnormal

24

fallopian tube epithelial cells to the ovary. WNT4 appears to be significantly amplified in

25

murine models of oviductal oncogenesis, as do select WNT receptors Frz2, 4, 5, and 8 [70]. -15-

1

Interestingly, a single nucleotide polymorphism in the WNT4 gene appears to have a

2

significant association with the incidence of ovarian carcinoma [83]. A genome wide

3

association study examining susceptibility loci for high-grade epithelial ovarian cancer noted

4

that amplification of the WNT4 locus is associated with increased incidence of disease [84].

5

RNA sequencing of a murine oviductal cell model lacking PTEN and characterized as high

6

grade oviductal carcinoma displayed significant upregulation of WNT4 and several other

7

genes associated with non-canonical WNT signaling [85]. This upregulation was similarly

8

validated in a transgenic model with conditional PTEN knockout, and was required for tumor

9

cell migration and colonization of the ovary in a β-Catenin-independent manner [86]. These

10

results were confirmed in human tumor microarrays and ovarian cancer cells lines, which

11

substantiate a pro-migratory role for WNT4 in ovarian cancer [86]. Interestingly, this study

12

also demonstrated that WNT4 is strongly expressed by the fallopian tube, implicating its use

13

as a marker of cell origin as well [86]. Taken together, these data suggest that the abundant

14

WNT4 in the fallopian tube may have key roles in stimulating the migration and invasion of

15

abnormal cells, thereby aiding the initiation of HGSOC. However, while a few treatment

16

methods involving canonical WNT signaling are currently under investigation, therapeutic

17

agents involving non-canonical signaling remain unstudied.

18

4.2 SDF-1

19

Stromal cell-derived factor 1 (SDF-1) and its receptor, CXCR4, play essential roles in

20

trafficking haemato- and lymphopoietic cells as well as stem and progenitor cells [87]. SDF-1

21

and CXCR4 are also expressed in ovarian cell lines and ovarian tumors, and SDF-1 is a

22

known component of follicular fluid [88]. Notably, in patients with ovarian cancer, an

23

increased expression of SDF-1 is associated with reduced overall survival while an increased

24

expression of CXCR4 is associated with reduced progression-free survival and overall

25

survival [88, 89]. The CXCR4 and SDF-1 interaction has been shown to stimulate invasion -16-

1

and proliferation in human ovarian cancer cell lines. CXCR4 upregulation is also associated

2

with disease progression and metastasis in human ovarian cancer specimens [90]. Ovarian

3

tumor initiating cells treated with a CXCR4 antagonist demonstrated a significant reduction in

4

migration [80]. The activation of ERK induced by the SDF-1 and CXCR4 interaction was

5

abrogated when cells were treated with the same antagonist [80]. Altogether, these data

6

suggest that SDF-1 and CXCR4 potentiate metastasis of ovarian cancer both from the

7

fallopian tube to the ovary as well as from the ovary to the peritoneum.

8

As such, inhibition of the SDF-1/CXCR4 signaling axis poses an interesting

9

opportunity for development of therapeutic agents. For example, treatment of ovarian cancer

10

cells with AMD3100, a selective CXCR4 antagonist, resulted in promotion of apoptosis and

11

inhibition of proliferation, migration, and invasion [91]. In addition, mifepristone, an FDA

12

approved chemical abortifacient, was able to suppress SDF-1 induced migration, invasion,

13

and adhesion of ovarian cancer cells [92]. Furthermore, pretreatment with mifepristone and

14

co-treatment with mifepristone and SDF-1 led to decreased tumor weight when compared to

15

treatment with SDF-1 alone [92]. However, more research is needed to determine if these

16

methods would be sustainable treatments for ovarian cancer.

17

4.3 ACTIVIN A

18

Activin A, a classic ovarian hormone belonging to the TGFβ superfamily, is composed

19

of two inhibin βA subunits, and signals through type I and type II activin receptors. In murine

20

oviductal cells, treatment with activin A resulted in induction of both EMT as well as increased

21

migration [93].

22

observation that treatment with activin A inhibitors decreased migration [93]. According to

23

clinical data in Oncomine, serous tumors had higher levels of activin A receptors and lower

24

levels of activin A signaling inhibitors when compared with normal ovarian samples [93].

Similar effects were noted in ovarian cancer cells with the additional

-17-

1

Additionally, higher expression of both the activin A type II receptor and subunit was

2

associated with decreased disease-free survival in patients [93]. Together, these data

3

suggest that ovarian activin A is capable of inducing migration in oviductal cells and

4

contributes to disease progression. However, a recent phase I clinical trial studying the

5

efficacy of the activin A inhibitor, STM 434, demonstrated no significant antitumor effect and

6

resulted in several adverse events [94].

7

4.4 NOREPINEPHRINE

8

Norepinephrine is a stress hormone secreted by adrenal glands and stored in various

9

tissues throughout the body. In a co-culture model of malignant human fallopian tube

10

epithelial cells and murine ovaries, imaging mass spectrometry showed that tumorigenic

11

murine oviductal epithelial cells, but not their normal counterparts, stimulated norepinephrine

12

secretion from the ovary [95]. Norepinephrine induced invasion in these cells as well,

13

suggesting it may be one of the factors driving the primary metastasis of ovarian cancer from

14

the fallopian tube to the ovary [95]. Interestingly, treatment of both fallopian tube and ovarian

15

epithelial cells with norepinephrine resulted in the down-regulation of several genes involved

16

in cell death, potentially allowing initial malignant cells to evade apoptosis [96].

17

As of late, norepinephrine has also been of interest at the clinical level. It is well known

18

that despite optimal first-line treatment (surgery and chemotherapy), around 70-80% of

19

patients with ovarian cancer will relapse with recurrent disease that is resistant to first-line

20

chemotherapeutic agents [97]. While this trend has not been entirely explained,

21

norepinephrine has been shown to stimulate DUSP1 expression which reduced platinum

22

agent induced apoptosis in ovarian cancer cells suggesting norepinephrine may mediate

23

chemoresistance [98]. Additionally, in a study of patients with epithelial ovarian cancer, use of

24

beta-adrenergic receptor blockers was associated with a 54% reduced chance of mortality

25

compared with non-users [99]. While similar studies have inspired clinical trials observing the -18-

1

effect beta-blockers may have on ovarian cancer patients without hypertension, little has

2

been done to investigate the effect of beta-blockers as a means of chemoprevention in high

3

risk patients [100]. Altogether, these data suggest that norepinephrine may significantly

4

mediate cancer progression and as such warrants further therapeutic consideration.

5 6 7

5. GENETIC ALTERATIONS AND THE TUMOR MICROENVIRONMENT Evidence suggests that mutations observed in HGSOC are often reflected in their

8

fallopian tube lesion precursors [21]. For example, a study examining ovarian tumors and

9

STIC lesions from individual patients determined that most had identical mutations in several

10

genes including TP53, PTEN, BRCA1, and BRCA2. As such, in the context of the fallopian

11

tube initiation model, it is likely that these mutations originated from tubal cells early in

12

tumorigenesis. Given the established roles for these genes in cell proliferation and motility, it

13

is also likely that cells from STIC lesions harboring select mutations may have aberrant

14

responses to the paracrine factors described in this review article.

15

cBioportal was used to examine patient data from The Cancer Genome Atlas

16

provisional dataset of high grade serous ovarian tumors along with the literature, which

17

implicated several pathways that are likely to alter paracrine or autocrine responses to the

18

ovarian microenvironment [101, 102]. Mutations affecting BRCA1, BRCA2, and PALB2 are

19

common to ovarian cancer (Table 1), with BRCA genes being the most common genetic risk

20

factor for HGSOC. These genes are known for their roles in the homologous recombination

21

DNA repair pathway. Given the known DNA damaging effects of several components of the

22

follicular fluid such as iron, AID, and various reactive oxygen species, it is important to

23

consider that FTE cells deficient in the homologous recombination pathway might incur more

24

damage or repair damage more slowly. Mutations to TP53 also modify cellular responses to

25

DNA damage and are present at 96-100% of HGSOC. As previously mentioned, fallopian

26

tube derived high-grade serous ovarian carcinomas carry early TP53 mutations. This is -19-

1

noteworthy because expression of mutant p53 in murine oviductal cells dramatically

2

increased expression of the progesterone receptor [103]. Treatment of p53-null murine

3

oviductal epithelial and p53-defective human fallopian tube epithelial cells with progesterone

4

induced necroptosis [104]. In a Trp53-/- murine model, similar necroptotic effects were

5

observed upon treatment with progesterone while inhibition of the progesterone receptor led

6

to double-strand break accumulation [104]. Activation of the necroptosis pathway could

7

explain the protective effect of progesterone against ovarian cancer. Interestingly, tumors

8

harboring

9

norepinephrine [95]. Cyclin E1 (CCNE1) is amplified in 12.97% of HGSOC patients (Table 1)

10

and has been found in early fallopian tube lesions, which is noteworthy considering

11

constitutive CCNE1 expression has been shown to induce chromosomal instability [105]. For

12

instance, CCNE1 amplification is associated with increased expression of ϒH2AX foci

13

suggesting that these cells may also accumulate damage in response to follicular fluid

14

differently than wild-type fallopian tube epithelial cells.

TP53

mutations

display

increased

invasive

potential

in

responses

to

15

In addition to the 87% of patients harboring TP53 mutations, mutations affecting PI3K

16

signaling were also prevalent (Table 1). 35% of patients had either mRNA and/or copy

17

number amplification of PIK3CA, while 17.4% had loss of PTEN. As both genes have been

18

linked to pathologic activation of PI3K signals, dysplastic tubal cells harboring these

19

mutations are likely to have abnormal responses to signals which exert their effects through

20

the PI3K pathway. Current models appear to substantiate this notion, as loss of PTEN results

21

in increased WNT4 signaling and a highly invasive phenotype [86]. Further, in the PAX8-Cre

22

transgenic murine models of oviductal derived HGSOC with loss of PTEN, mutant TP53, and

23

either mutant BRCA1 or BRCA2, these tumors formed rapidly and when animals were

24

ovariectomized, they failed to disseminate into the peritoneal space [82]. Ovarian cancer

-20-

1

patients also displayed frequent loss of NF1, a negative regulator of RAS signaling, which

2

may also have significant effects on cellular responses involving downstream MAPK signaling

3

networks including PI3K (Table 1). As activin A, estrogen, prolactin, and SDF1 are dependent

4

on RAS/PI3K signaling to exert their pro-migratory functions, the contributions of these

5

mutations to tumor cell recruitment warrant further study. Interestingly, we also observed

6

frequent mRNA downregulation of SMAD2, a critical mediator of activin and TGFβ signaling

7

(Table 1), both of which are known mediators of the tumor microenvironment contributing to

8

several hallmark features of tumorigenesis including proliferation, migration, epithelial to

9

mesenchymal transition, and immune evasion [106, 107]. As such, should loss of SMAD2

10

occur early in precursor lesions, this may have substantial ramifications in responses to

11

TGFβ family ligands in the follicular fluid or ovarian secretions.

12

In summary, genomic alteration to these and many genes in dysplastic fallopian tube

13

epithelial cells may lead to extensive signal reprogramming, subsequently affecting the

14

interactions of said cells with paracrine signals from the surrounding fallopian tissue or ovary.

15

Though potentially important to our understanding of HGSOC pathogenesis, how such

16

mutations alter the interaction of tubal cells with the local tumor microenvironment is largely

17

unstudied. Hence, further examination is necessary to better evaluate the early effects of

18

these and other mutations, particularly regarding the design of early intervention strategies.

19 20 21 22

6. CONCLUSION Several factors in both fallopian tube and ovarian secretions along with those

23

contained in follicular fluid may be responsible for the transformation and dissemination of

24

dysplastic fallopian tube epithelial cells to the ovary aiding in the establishment of ovarian

25

cancer. These factors range in effect which include transformation, proliferation, and

26

migration. While some of these factors are always present in the tissues, others are acquired -21-

1

during malignancy. A combination of mutations common to ovarian cancers can ultimately

2

lead to pathway modifications capable of disease development (Figure 2). For instance, one

3

of the earliest mutations found in SCOUT lesions is a loss of PAX2, which leads to increased

4

susceptibility to estrogen signaling. Since estrogen signaling can then stimulate cellular

5

damage and resistance of apoptosis, this loss of PAX2 can allow for perpetuation of

6

damaged cells. Other common mutations found in early lesions include alterations to p53 and

7

loss of PTEN. Lesions featuring these genotypic alterations are known to have increased

8

WNT4, which results in increased capacity for metastasis and invasion. Additionally, mutation

9

of p53 may alter the response of fallopian tube cells to progesterone to induce necroptosis.

10

The surrounding norepinephrine secreted from ovarian tissue is capable of stimulating

11

migration of these cells as well. In addition to the aforementioned mutations and impactful

12

factors, follicular fluid is released into the environment surrounding both the fallopian tube

13

and ovary on a monthly basis from the age of puberty (10-14 years) until menopause (45-55

14

years). Ultimately, a combination of events such as the ones described can transform benign

15

fallopian tube epithelial cells into lesions capable of further progression, metastasis, and

16

invasion of the ovary allowing for the formation of overt tumors. Importantly, while many of

17

the components mentioned in this review have provided potential points of therapeutic

18

intervention, further research on these various signals is required in order to fully elucidate

19

their role in the initiation and progression of ovarian cancer in order to develop effective

20

chemotherapeutic agents.

21

-22-

1 2 3

Figure 1. Signals of Interest in Ovarian Cancer Development

4

exist throughout the fallopian tubes and are regularly exposed to several potentially

5

oncogenic factors. These factors stem from secretions, from fallopian tube and ovarian

6

epithelium, and follicular fluid that is released upon follicle rupture during the reproductive

7

cycle. They can be protective or transformative, and stimulate proliferation or metastasis of

8

fallopian tube epithelial cells.

9 10 11 12 13

Serous tubal intraepithelial carcinomas (STICs) and other precursor lesions of ovarian cancer

Figure 2. Changes in Ovarian-Fallopian Tube Communication from Normal Fallopian Tube Epithelium to Intraepithelial Carcinoma Before the occurrence of any transformational events, normal fallopian tube epithelium

14

secretes several factors that likely play a role in disease development. If the epithelium forms

15

a SCOUT lesion, a serous outgrowth typically PAX2 null, it then expresses increased

16

estrogen receptor leading to increased estrogen signaling. If cells undergo loss of PTEN, they

17

express higher levels of WNT4 and are susceptible to the proliferative and metastatic effects

18

of norepinephrine. Mutation of p53 leads to the development of a signature lesion. In p53 null

19

signature lesion cells, progesterone (P4) can protectively induce necroptosis. Combinations

20

of these mutation and factor-induced transformations promote development of serous tubal

21

intraepithelial carcinomas (STICs). STICs are considered malignant and are capable of

22

dissemination to and invasion of the ovary.

23

-23-

1 2 3 4 5

Table 1. The frequency of genomic alterations in key signaling genes in the TCGA Firehose Legacy ovarian serous cystadenocarcinoma cohort cBioportal was utilized to analyze ovarian serous cystadenocarcinoma patient data from The

6

Cancer Genome Analysis Firehose Legacy patient data set (N = 606) according to the

7

original references [101, 102]. Protocols for sequencing analysis of TCGA dataset can be

8

located here: https://tcga-data.nci.nih.gov/tcga/. Genetic analyses were conducted using only

9

samples with mutation data (N = 316).

10

-24-

1

ACKNOWLEDGEMENTS

2

This work is supported by grant CA240301 and startup funds provided by the

3

University of Illinois Urbana-Champaign. We would also like to acknowledge BioRender for

4

enabling the creation of the figures featured in this work.

5

CONFLICT OF INTEREST STATEMENT

6

The authors have no conflicts to disclose.

7 8

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Gene

Total Alterations

Mutation

Amplification

Deletion

mRNA Upregulation

mRNA Downregulation

Multiple Alterations

TP53

88.29%

87.03%

-

0.32%

-

-

0.95%

PIK3CA NF1 CCNE1 SMAD2 PTEN BRCA2 PALB2 BRCA1

35.44% 26.58% 26.27% 20.33% 17.41% 11.71% 11.08% 8.23%

0.63% 1.9% 0.32% 3.48% 1.27% 3.8%

18.35% 12.97% 1.1% 0.63% 1.9% 0.63% 0.32%

0.95% 1.1% 0.63% 0.95% 1.58%

6.65% 0.63% 2.22% 1.65% 2.85% 3.48% 0.95% 0.95%

14.24% 2.85% 12.64% 7.91% 1.27% 8.23% 0.95%

9.81% 8.86% 8.23% 3.85% 5.06% 0.63% 0.63%

HIGHLIGHTS •

Ovarian cancer is the most lethal gynecologic malignancy

•

Fallopian tube epithelium is a source for high grade ovarian cancer

•

New analysis of transformation and metastasis can improve diagnosis and treatment

CONFLICT OF INTEREST STATEMENT The authors have no conflicts to disclose.