Background
Transforming growth factor beta (TGFB) superfamily members regulate a variety of biological processes in a contextually dependent manner via the interaction with membrane associated TGFBR1/TGFBR2 serine/threonine kinase receptor complexes and downstream SMAD proteins [
1]. A growing body of research documents critical roles of TGFB signaling in reproductive development, function, and dysfunction [
2,
3]. TGFB signaling is known for its tumor suppressive function, particularly in epithelial cancer cells [
4,
5]. However, TGFB signaling also promotes cancer progression through modulating tumor cell invasion and metastasis [
6]. Recently, we have shown that TGFB pathway activation plays a crucial role in pancreatic tumor initiation, through its capacity to induce Acinar-to-Ductal metaplasia, providing a favourable environment for KRAS
G12D-dependent carcinogenesis [
7].
Disruption of genes encoding TGFB superfamily signaling mediators [e.g., inhibin alpha (
Inha), bone morphogenetic protein (BMP)-responsive SMADs (i.e.,
Smad1/5), and BMP type 1 receptors (i.e.,
Bmpr1a and
Bmpr1b)] results in the development of sex cord-stromal tumors [
8‐
10]. Studies have revealed tumorigenic function of activin-responsive SMAD3 and tumor suppressive roles of inhibins and BMPs in the gonad [
8‐
11]. A cornerstone study has demonstrated that nearly all adult granulosa cell tumors (GCTs), a major subtype of sex cord-stromal tumors, harbor a somatic mutation of forkhead box L2 (
FOXL2; 402C→G; C134W) [
12]. However, the specific role of
FOXL2 mutation and its associated tumorigenic signals in GCTs remain to be clarified. Therefore, it is imperative to define the mechanism underlying the initiation and progression of ovarian GCTs, with an ultimate goal of developing new therapeutic strategies for this type of poorly characterized tumor.
As the functional unit of the ovary, a follicle consists of granulosa and theca somatic cells and a germ cell, the oocyte. Through secretion of paracrine growth factors, the oocyte regulates multiple functions of granulosa cells including, but not limited to, apoptosis, proliferation and differentiation, steroidogenesis, and metabolism [
13,
14]. It is noteworthy that dysregulation of phosphoinositide-3-kinase (PI3K)-AKT signaling in the mouse oocyte impairs normal ovarian function and causes ovarian GCT formation [
15]. In an early report, we have shown that constitutive activation of TGFB signaling in mouse granulosa cells promotes ovarian tumorigenesis [
16]. To further explore the tumorigenic function of dysregulated TGFB signaling in distinct cellular compartments of the ovary, we created a mouse model that harbors constitutively active TGFBR1 using growth differentiation factor 9 (
Gdf9)-iCre known to be expressed in the oocyte.
Discussion
GCTs are the major type of sex cord-stromal tumors, and both genomic and genetic factors are involved in the pathogenesis of these tumors [
34,
35]. Human GCTs can be divided into adult and juvenile types; the former represents more than 90% of all GCTs and mainly occurs in perimenopausal or postmenopausal women around 50-54 years of age [
36]. The Juvenile GCTs are relatively rare and mainly occur in prepubertal girls [
37]. Although the 5-year survival rate in stage I patients is generally high, a poor prognosis occurs in patients with advanced stages of disease [
38‐
40]. Of note, human adult GCTs have a high risk of recurrence [
41], leading to resistance to chemotherapy and death. Because of the rarity of this category of tumors, mouse models are beneficial to study the etiopathology of these tumors.
Several reports using genetically modified mouse models highlight the importance of TGFB superfamily signaling components, such as INHA, SMAD3, SMAD1/5, and BMPR1A/BMPR1B, in sex cord-stromal tumor development [
8‐
11,
42,
43]. A causal link between the mRNA expression levels of TGFBR1 and the development of GCTs in humans has not been established [
43]. Growing evidence supports that activation of TGFB/activin signaling may represent a driving force of GCT development in mice [
44]. It has also been found that TGFB/activin signaling is active in human GCTs [
32,
45]. Notably, nearly all adult GCTs bear a somatic missense mutation of
FOXL2 C→G (C134W) [
12], which may alter activin/TGFB and BMP signaling activity and the proliferation and differentiation status of granulosa cells [
46‐
48]. In an effort to define the role of aberrant activation of TGFB signaling in the pathogenesis of ovarian tumors, we utilized a mouse model harboring constitutively active TGFBR1. Our results corroborate the role of TGFB signaling in ovarian tumorigenesis evidenced by the finding that constitutive activation of TGFB signaling in ovarian somatic cells leads to the formation of ovarian malignancies that phenocopy GCTs in several perspectives [
16]. Of note, sustained activation of TGFBR1 led to the phosphorylation of SMAD2/3, which are downstream signaling elements shared by TGFBs and activins. Therefore, our results do not discount the role of activin signaling in GCT development. Indeed, our early studies have shown that activin signaling is important in promoting ovarian sex cord-stromal tumor development [
11,
42]. Collectively, our approach to manipulate TGFBR1 activity and SMAD2/3 activation represents a valuable tool to study ovarian GCT development.
Oocyte-somatic cell communication is critical for follicular development. Through the secretion of growth factors, some of which are TGFB superfamily proteins, oocytes regulate folliculogenesis in a bi-directional paradigm [
13,
14]. The involvement and potential role of oocyte-granulosa cell regulatory loop in sex cord-stromal tumor development are poorly understood. A recent study has provided functional evidence that constitutive activation of PI3K-AKT signaling in the oocyte promotes GCT formation [
15], suggesting that dysregulation of major growth regulatory pathways in the oocyte impacts granulosa cell growth and differentiation. Genetic evidence suggests that SMAD4-dependent canonical TGFB signaling in oocytes is largely dispensable for female fertility [
49]. However, it has not been determined whether unopposed TGFB signaling in the oocyte is detrimental to ovarian development and function. In the current study, ovarian tumors developed in mice harboring
TGFBR1
CA and
Gdf9-iCre. By performing histological and molecular analyses, we demonstrated that the ovarian neoplasms were sex cord-stromal tumors which expressed granulosa cell markers including FOXL2, INHA, and FOXO1. An elegant review by Pitman and colleagues suggests that premature loss of ovarian germ cells may promote the formation of ovarian neoplasms [
33]. However, the tumor phenotypes appear to be associated with the type of mutations and the onset of oocyte loss [
33]. In this study, the specific cause of follicle/oocyte reduction and its potential involvement in ovarian tumor development remain unknown. The observation that overactivation of TGFBR1 using
Gdf9-iCre did not increase oocyte apoptosis suggests that the reduction of follicle/oocyte numbers in TGFBR1-CA
G9Cre mice may be associated with the disruptive effect (e.g., impairment/destruction) of tumor formation and development.
The development of GCTs in TGFBR1-CA
G9Cre mice begs the question of how overactivation of TGFBR1 links to ovarian tumor formation. As a first step, expression of
Gdf9-iCre was verified in our model. Our X-gal staining showed predominant Cre activity in the oocyte. Somewhat unexpectedly, we also observed minor sporadic signals in some follicles. Of note, in TGFBR1-CA
G9Cre mice,
Gdf9-iCre was transmitted maternally due to the extremely low efficiency to generate
TGFBR1
CA flox/+;
Gdf9-iCre mice using male breeders (i.e.,
Gdf9-iCre males) to transmit
Gdf9-iCre. In our breeding strategy,
Gdf9-iCre females were crossed with
TGFBR1
CA flox/+ male mice to produce
TGFBR1
CA flox/+;
Gdf9-iCre females. The reason for the inability of obtaining
TGFBR1
CA flox/+;
Gdf9-iCre mice using males to transmit
Gdf9-iCre was not clear. Since constitutively active TGFBR1 in ovarian somatic cells is a strong driver of ovarian GCTs in mice [
16], it was possible that even low to negligible Cre activity in a subset of granulosa cells might be permissive for
TGFBR1
CA activation, leading to GCT development. Further supporting the contribution of overactivation of TGFBR1 in granulosa cells to ovarian tumorigenesis in the TGFBR1-CA
G9Cre model, mice harboring
TGFBR1
CA and
Zp3-Cre, which is expressed in growing oocytes, but not non-growing oocytes of primordial follicles [
19], did not develop ovarian tumors, regardless of the parental origin of Cre transmission (Y. Gao and Q. Li, unpublished observation). We previously found that ovarian GCTs induced by overactivation of TGFBR1 in granulosa cells express higher levels of
Gli1 and
Gli2 transcription factors and lower levels of
Tgfbr3 compared with normal ovaries [
16]. Interestingly, a similar transcript expression pattern of these genes was found in the ovaries of TGFBR1-CA
G9Cre mice. These findings collectively suggest that ovarian tumor formation in TGFBR1-CA
G9Cre mice may be attributable, at least partially, to overactivation of TGFBR1 in the granulosa cell compartment. Since the
TGFBR1
CA is tagged with HA, we have tested the utility of a number of commercially available antibodies directed to HA in immunohistochemical/immunofluorescence applications, with the aim of identifying malignant granulosa cells harboring TGFBR1 overactivation. Although some antibodies performed reasonably in western blotting analysis, none of them generated reproducible and convincing results in immunohistochemical/immunofluorescence assays. Future work using
Rosa26/
TGFBR1
CA/
Gdf9-iCre mice may help elucidate the cellular origin of TGFBR1 overactivation and its contribution to ovarian GCT formation. It is worthwhile mentioning that a potential contribution of overactivation of TGFBR1 in the primordial oocyte or in both primordial oocytes and granulosa cells to GCT development could not be excluded. Further clarification of this question relays on the technical capability of specifically manipulating TGFB signaling in the oocyte of primordial follicles.
In summary, this study has created a mouse model of GCTs with defined disease onset that can be further exploited to study the role of TGFB signaling in ovarian tumor development. Of note, our studies were performed using mice and extrapolation of the findings to human GCTs needs further investigation. Identification of molecular markers for GCTs would benefit early diagnosis and treatment. To date, there are no reliable biomarkers for GCTs, although serum levels of estradiol, inhibin, and AMH have been extensively investigated and appear to correlate with GCTs in some cases [
50]. Ongoing studies are to identify molecular signatures of GCTs in our model during tumor initiation and development.