Cellular models of development of ovarian high‐grade serous carcinoma: A review of cell of origin and mechanisms of carcinogenesis

Abstract High‐grade serous carcinoma (HGSC) is the most common and malignant histological type of epithelial ovarian cancer, the origin of which remains controversial. Currently, the secretory epithelial cells of the fallopian tube are regarded as the main origin and the ovarian surface epithelial cells as a minor origin. In tubal epithelium, these cells acquire TP53 mutations and expand to a morphologically normal ‘p53 signature’ lesion, transform to serous tubal intraepithelial carcinoma and metastasize to the ovaries and peritoneum where they develop into HGSC. This shifting paradigm of the main cell of origin has revolutionarily changed the focus of HGSC research. Various cell lines have been derived from the two cellular origins by acquiring immortalization via overexpression of hTERT plus disruption of TP53 and the CDK4/RB pathway. Malignant transformation was achieved by adding canonical driver mutations (such as gain of CCNE1) revealed by The Cancer Genome Atlas or by noncanonical gain of YAP and miR181a. Alternatively, because of the extreme chromosomal instability, spontaneous transformation can be achieved by long passage of murine immortalized cells, whereas in humans, it requires ovulatory follicular fluid, containing regenerating growth factors to facilitate spontaneous transformation. These artificially and spontaneously transformed cell systems in both humans and mice have been widely used to discover carcinogens, oncogenic pathways and malignant behaviours in the development of HGSC. Here, we review the origin, aetiology and carcinogenic mechanism of HGSC and comprehensively summarize the cell models used to study this fatal cancer having multiple cells of origin and overt genomic instability.


| High-gradeserouscarcinomaoftheovary, peritoneum and fallopian tube
Epithelial ovarian cancer (EOC) is one of the most common malignant tumours in women, with approximately 21 750 new cases and 13 940 deaths estimated in the United States in 2020. 1 The 5 year survival rates have been <45% for many years, 2 which indicate that either the prevention or the treatment has not improved significantly over the past decades.
Among the different histological types of EOC, high-grade serous carcinoma (HGSC) is both the most prevalent and most fatal type, accounting for 30%-60% of cases and 70%-80% of the mortalities. 3 The prevalence and high mortality are mainly due to the difficulty of early diagnosis and propensity for recurrence because of resistance to chemotherapeutic agents. [4][5][6] Consequently, EOC is the 7th most common cause of cancer-related deaths among women in the world. 1 Obvious impediments to progress include unclear aetiology, ambiguous tissue of origin and unknown mechanism of malignant transformation. [7][8][9] Besides the ovary, HGSCs are occasionally found in the fallopian tube and peritoneum as the main lesion. 10 These extraovarian HGSCs show identical characteristics to the ovarian counterpart, including genetic, molecular and histological features and clinical behaviours, 11 indicating they are the same disease of different localization.

| OvarianandtubaloriginofHGSC,a debated and shifting paradigm
Ovarian surface epithelium (OSE) or cortical inclusion cysts have long been considered to be the orthodox origin of ovarian HGSC. 12 This dogma was challenged by findings showing that precursor lesions of HGSC termed 'p53 signature' and serous tubal intraepithelial carcinoma (STIC) were exclusively found in the epithelium of the fallopian tube, especially at the fimbria part, but not on the ovary. [13][14][15][16] This shifting paradigm was further reinforced by intensive histopathological analyses, clonality assays of the driver mutations and the whole genome [17][18][19] of surgical specimens as well as by genetic manipulation in cellular and transgenic mouse models recapitulating the transformation from fallopian tube epithelial cells (FTECs). [20][21][22] The data from these various investigations point to the secretory cells in fimbrial epithelium as the main cell of origin of HGSC. [23][24][25] However, this conclusion does not suggest that all HGSCs have a fallopian tube origin. Nostalgic studies involving cellular transformation and a genetic-engineered mouse model (GEM) keep providing pieces of evidence that HGSC can also arise from OSE. [26][27][28] The current consensus is that HGSC can arise from both OSE and the fallopian tube, with the fallopian tube epithelium (FTE) as the major origin. 27,29

| Mechanismoftransformation:Ovulationdriven mutagenesis and clonal expansion with loss of progesterone protection
More and more epidemiological studies have supported the theory of incessant ovulation as the cause of HGSC. 30,31 The hypothesis that ovulation is associated with ovarian cancer was first raised in 1971 by MF Fathalla. 32 In numerous large-scale epidemiological studies, the number of ovulations was associated with an increased risk of ovarian cancer in a dose-dependent manner. [33][34][35][36] Factors that reduce the number of ovulation cycles, such as pregnancy, lactation and use of oral contraceptives, showed a protective effect on HGSC. 32,37 The underlying mechanisms of carcinogenesis caused by incessant ovulation were explored under different assumptions. The endocrine mechanism stating a carcinogenic role of gonadotropins and oestradiol, which peak during ovulation and exert a transformation effect, 37-39 was refuted by a study showing that FTECs did not proliferate or display increased DNA damage in response to either oestrogen or follicle-stimulating hormone/luteinizing hormone. 31 Alternatively, the tear-and-repair hypothesis proposed by Fathalla 32 states a repetitive proliferation of the OSE after ovulation-induced wounding, 40 which increases DNA damage and may enhance the transformation. 41 It was later noted that without a tearing, fallopian tube fimbria, bathed by follicular fluid (FF) during oocyte catch up, is also vulnerable to ovulation injury, such as inflammation and DNA damage. [42][43][44][45] Eventually, the focus turned to the contents in the FF that bath both the ovulatory wound and FTE after ovulation. FF contains high concentrations of growth factors, proteinases involving coagulation cascades and others, extracellular proteins, hormones, immune agents and reactive oxygen species (ROS). 46-49 Among them, ROS causes tissue injury and DNA double-strand breaks on the epithelium of fallopian tube fimbria and was regarded as a mutagen in FF, 48,50,51 and some growth factors such as IGF2 cause clonal expansion and transformation of immortalized FTECs. 48,51,52 More than the DNA damage, FF also exerts stem cell activation and clonal expansion activity on FTECs. FF contains abundant insulin-like growth factor (IGF)-axis proteins, including IGF-binding protein 2/6, IGF1/2 and the IGF-binding proteolytic enzyme, pregnancy-associated plasmatic protein A (PAPP-A). The expression levels of these proteins increased with the growth of the ovarian follicle. 53 After ovulation, the IGFBP-cleavage enzyme PAPP-A is activated upon tethering to the membrane of FTECs. IGF2 is then released and binds to the adjacent mechanism of HGSC and comprehensively summarize the cell models used to study this fatal cancer having multiple cells of origin and overt genomic instability. membrane receptor IGF-1R and activates the AKT/OCT2/ NANOG and AKT/mTOR pathways. These signals of stemness activation and clonal expansion, on the one hand, repair the injury caused by ovulation, and on the other, lead to expansion of cancer initiation cells and malignant transformation. 52 Thus, a regeneration mechanism is reserved in the ovarian follicle, which is timely and locally activated after ovulation. However, the cost is carcinogenesis of the exposed epithelium of the fimbria and the ovary, a justifiable price following the priorities of reproduction and evolution.
In addition to ovulation, the backflow of menstrual blood or retrograde menstruation may also promote development of ovarian cancer. The risk of ovarian cancer decreased after the retrograde route was blocked by tubal ligation. [54][55][56] Iron metabolites in menstrual blood exposed to the FTE and ovary may exert the Fenton reaction upon iron oxides by interacting with H 2 O 2 released from ovulation. It was reported that iron-transporter protein transferrin, via its receptor TfR1, facilitated formation of DNA double-strand breaks in FTE. 57 Moreover, haemoglobin from retrograded menstrual blood readily quenched the excessive ROS released from ovulation and rescued the exposed FTE from the ROS-induced apoptosis. The surviving FTE under this tolerable ROS stress still accumulates DNA double-strand breaks and proceeds to transformation. 48 Therefore, ovulation and retrograde menstruation produce a repeated tolerable Fenton reaction on FTE and OSE, which may promote development of ovarian cancer. 58,59 Given that both ovulation and retrograde menstruation are almost regular events 60 but the prevalence of STIC (<1%) 61 65 Interestingly, PR is downregulated in the most majority of EOCs. 66 Two polymorphisms at the PGR gene contribute to ovarian cancer susceptibility. 67 Thus, either intrinsic or extrinsic progesterone protects the development of HGSC at early stage, and loss of PR may be a necessity for the carcinogenesis induced by ovulation and retrograde menstruation.

| Molecularpathwaysinvolvedinthe developmentofHGSCanditsprecursorsinthe fallopian tube
Almost all HGSCs and their tubal precursor STICs harbour TP53 mutations, 25,68-70 which are considered to be the first step in the transformation. 71 The initial TP53 mutation lesion, p53 signature, is a cluster of histologically normal tubal secretory cells with accumulation of mutant p53 in the nucleus, which was estimated to occur about 10 years after the first ovulation. 64 DNA double-strand breaks frequently occur in p53 signatures, indicating that it may be induced by ovulation-and retrograde menstruation-related ROS.
A more severely transformed tubal epithelial lesion is called STIC, which retains the expression of oviduct secretory cell marker PAX8, TP53 mutation and DNA damage, 72 and acquires high proliferative activity, cell atypia and loss of cellular polarity. After acquiring metastatic properties, STICs spread to peritoneal organs, including the ovary and peritoneum and becomes clinically evident HGSC. 73 Interestingly, while STIC has a propensity for intraperitoneal metastasis, it rarely invades deeply into the lamina propria to grow overt fallopian tube cancer. Thus, STIC is found either as an in situ carcinoma or microinvasive carcinoma. Clinically, HGSC mostly presents in the ovary as the primary site rather than in the fallopian tube.
We reason that the stroma of fallopian tube must have evolved a mechanism to resist the implantation/invasion of embryo to prevent the fatal ectopic pregnancy. The same mechanism also impedes the invasive growth of STIC. Among them, TP53 mutation is the earliest universal hit. [76][77][78] In addition to cell cycle control, mutant p53 proteins may acquire gain of function (GOF) activity. These proteins can interact with new DNA targets and protein partners, promoting genomic instability, proliferation, invasion, metastasis, inflammation, angiogenesis and chemoresistance. 79 Clinical data have shown that the prognosis of HGSC patients with GOF p53 mutations was poorer than that of patients with loss of function p53. 80 Mutation or amplification of the RB pathway genes occurs in 2/3 of cases and early after TP53 change. This early dual disruption of p53 and Rb pathways underscores the DNA copy number variation and chromosomal instability phenotypes present in tubal precursor lesions early in HGSC development. 74 Additionally, BRCA1/2 and PTEN mutations also have been found in STIC lesions, 19

| CELLMODEL SUS EDFORTHES TUDY OFTHEG ENE TICALTER ATI ONAND MECHANIS MOFHG SCDE VELOPMENT
Given the shifting paradigm of cell origin, cell lines derived from the FTE with different severities of oncogenic alterations were established to explore the molecular mechanism of cell transformation, biomarkers for early detection and prevention methods. Herein, we summarize the immortalized and transformed cell lines derived from the oviduct of humans and mice and their applications in this rapidly evolving field of research.

| HumanHGSCcelllines
Cancer cell lines used as a tumour model in vitro have had a profound impact on cancer research and greatly promoted the development of new biomarkers and targeted cancer therapies. [84][85][86] However, before the molecular classification and binary stratification system was established, EOC cells could not be discerned in studies. In addition, misidentification and cross-contamination of some cell lines have also hindered research progress. 87 The consequence has been a prolonged delay in discovery of targeted therapies and specific biomarkers in ovarian cancer. Another problem is that some ovarian cancer cell lines do not match the characteristics of homologous tumours. 88,89 Given the knowledge of the molecular characteristics of different EOC, particularly the unique features of HGSC, that is the universal TP53 mutation and profound DNA copy variation, re-

Cell name Morphology
Original

| Immortalizedfallopiantubesecretorycelllines
To explore the pathogenesis of a cancer, cell lines deriving from the  Table 2 lists these immortalized FTECs, methods of immortalization and their genetic alterations. These immortalized FTECs showed phenotypes of fallopian tube secretory cells expressing markers, such as PAX8 and WT1, 116 and do not express ciliated cell markers, such as FOXJ1. 117 The majority of these cells do not grow colonies in soft agar, and some showed limited anchorage independent growth (AIG), suggesting evolution of early transformed clones. 96

| Invivoandinvitrotransformedcellmodels
With immortalized FTECs available, driver mutations have been introduced and examined for their transformation capability. For example, inactivation of Rb but not Brca1, together with Trp53 inactivation, was found to be sufficient for mouse OSE transformation. 118,119 To explore the role of CCNE1, which is a negative regulator of RB and frequently amplified early in HGSC development, CCNE1 was overexpressed in TP53 R175H -mutated FT282 cells to derive the The results indicated that downregulation of CNN1 was necessary for anoikis survival and cell transformation, the essential step for HGSC metastasis. 132 The YAP transcription activating protein, which mediates growth-suppression signals downstream of various biological and environmental cues, was tested in FT194 cells. Xenograft of FT194-YAP cells resulted in slow-growing small subcutaneous tumours. As a comparison, FT194 carrying S127A mutant YAP resulted in rapid growing and larger tumours, which was consistent with the phenotype of HGSC. 81,133 At present, YAP signalling activation is the only known independent oncogene in non-viral oncoproteins that have been shown to promote tumour formation by immortalizing FTSECs.  Table 3 and Figure 1 present the above-mentioned tumourpromoting and tumour-suppressing genes/molecules that are important for the development of HGSC and have been tested in the immortalized FTECs in vitro and in vivo.

| Immortalizedmurineoviductepithelial cellsandOSEcells
Unlike human cells that require transgenes, such as hTERT, to over-

| Limitationsandimprovementsofcurrent cell models
On the one hand, the molecular and histological characteristics of the currently constructed cancer cell lines were inconsistent with the original tissues. 146

| CON CLUS ION
With more in-depth research on the origin of ovarian HGSC, ovaries and FTE cells are increasingly considered to be the main origins of this cancer, and more cell lines and animal models have been constructed in the process. In this review, we summarized the currently available

CO N FLI C TO FI NTE R E S T
The authors declare that there is no potential competing interest.

AUTH O RCO NTR I B UTI O N S
TC and JM conceived the structure of manuscript and revised the manuscript. JM and HT made the figures and table. HH, CH and NW completed a large number of preliminary studies. YL and WZ revised the manuscript. All authors approved the final manuscript.

DATAAVA I L A B I L I T YS TAT E M E N T
Data sharing is not applicable to this article as no new data were created or analysed in this study.