Under physiologic conditions, the granulosa cell is regulated by reproductive hormones and growth factors in an endocrine, paracrine and autocrine manner 
. Any disruption in the regulatory pathways that function during normal ovarian development, folliculogenesis, and ovulation may result in uncontrolled granulosa cell proliferation and lead to malignant transformation. Several factors and cellular signaling pathways have been implicated in the initiation and progression of GCT. For example, studies with mutant mouse models show that a deficiency in α-inhibin expression contributes to initiation of GCT 
. However, as patients with GCTs generally have high levels of circulating inhibins 
, the role of inhibin in the development of GCTs in humans requires further evaluation. More recent studies with mouse models suggest that the loss of PTEN or activation of kRas and concomitant activation of Wnt/β-catenin signaling contributes to the initiation and progression of GCT 
. Despite the compelling GCT phenotypes, the relevance of these pathways in the initiation of juvenile or adult human GCTs has not been established. Recent somatic gain-of-function studies have shown that mutations in the transcription factor FOXL2 may be an important biomarker for the diagnosis of human ovarian GCTs 
. Nevertheless, the mechanisms underlying the biologic activities and role of FOXL2 in the initiation, progression, recurrence and metastasis of ovarian GCT remain unknown. Our present study shows that the ErbB family of tyrosine kinase receptors is expressed in human granulosa tumor tissues and two human GCT cell lines. We observed that TGFα, via EGFR, promoted proliferation of KGN and COV434 cells and facilitated KGN tumor cell migration. Our data also indicate that multiple pathways mediate the stimulatory effects of TGFα on proliferation of the KGN GCT cells. Although the function of the ErbB family on ovarian follicle development, ovulation, and ovarian epithelial tumorigenesis have been extensively studied 
, to our knowledge, this is the first direct evidence showing the function of EGF family of ligands, specifically TGFα, on GCT cell growth and possibly in GCT progression.
Mutation, overexpression of receptors, or constitutive activation of the downstream pathways of ErbB receptors are associated with many diseases, including ovarian cancer 
. Expression of ErbB receptors on normal ovarian cells and on epithelial ovarian cancer cells has been well studied 
. On the other hand, the results from two reports on the expression of ErbB family proteins in human GCTs are inconsistent. Furger et al. 
showed that ErbB2 is expressed in the COV434 granulosa tumor cell line and in 6 of 12 human GCT tissue samples. Conversely, a more recent immunohistochemical study of 40 GCT cases reported that ErbB2 was undetectable in all 40 cases 
. The reason for these differences is not clear since both studies used an ErbB2 antibody from the same source. Our study using another validated antibody showed that ErbB2 was present in KGN and COV434 cells and 5 of 5 patient samples. Furger et al. 
also showed that 2 of 12 patient tumor samples expressed ErbB3, and 10 of 12 expressed ErbB4. However, Leibl et al. 
reported that EGFR was present in 65% of the cases, and positive reactions for ErbB3 and ErbB4 were observed in 45% and 58% of the cases, respectively. Our present results in 5 human GCT cases and 2 GCT cell lines showed positive staining for ErbB3 and ErbB4. Based on these findings, it seems likely that GCTs will express multiple members of the ErbB family of receptors.
We observed that the expression patterns of ErbB receptors in the two GCT cell lines were different, with the COV434 cell line expressing higher levels of ErbB3 and ErbB4. Despite these differences TGFα stimulated proliferation of both cell lines. It is possible, however, that these two cell lines may respond differently to other members of the EGF family of ligands (e.g., heparin-binding EGF-like growth factor, amphiregulin, betacellulin, epiregulin, neuregulin) based on increased expression of ErbB3 and ErbB4 in COV434 cells. Evidence in other systems indicates the EGF family of ligands is capable of activating distinct ErbB receptors, signaling pathways, and biologic responses 
. Additional experiments are warranted to test this possibility. Our data also showed that TGFα and EGF are expressed in both GCT cell lines. Expression of both receptors and ligands in GCT cells suggests that autocrine and/or paracrine mechanisms may be involved in TGFα regulation of GCT cell growth. It is well-known that activation of EGFR by TGFα or EGF leads to a rapid internalization of these receptors. The internalized EGFR is transported to early endosomes where receptor-ligand complexes are sorted for either degradation or recycling to the cell surface 
in the present study clearly show that the total EGFR level significantly decreased in KGN cells 6 hours after TGFα stimulation (), suggesting that the internalization and recycling pathways are active in KGN cells.
Our current study demonstrates that TGFα stimulates the proliferation of KGN and COV434 cells. These findings are somewhat inconsistent with previously published results. Imai et al. 
reported that exogenous EGF did not influence KGN cell proliferation. Unfortunately, it is not possible to directly compare the approaches in this study with the previous report due to a paucity of experimental detail in the previous report. One explanation for this discrepancy may be that TGFα is a more potent mitogen than EGF in KGN cells. It is also possible that in some circumstances, TGFα and EGF have different functions on certain cells 
. Moreover, we observed that stimulation of KGN cells with EGF did not promote KGN cell proliferation in serum-free medium; in contrast TGFα stimulated cell growth under serum-free conditions (Supplemental ). However, if the culture medium was supplemented with 10% of serum, both TGFα and EGF significantly stimulated KGN cell proliferation. Thus, other hormones or cytokines may also be required for EGF to optimally stimulate KGN cell proliferation. It seems that the presence of these unknown factor(s) may increase the sensitivity of KGN cells to TGFα and EGF stimulation.
GCTs are able to metastasize to many organs 
, but the mechanisms underlying GCT metastasis are largely unknown. It seems likely that hormones and growth factors may contribute to GCT metastasis. A recent report showed that KGN cells were capable of metastasis after implantation into nude mice 
. The metastasis of the KGN cell tumor developed slowly and mimicked the characteristics of GCT metastasis reported previously 
. We found that TGFα had a profound effect on the morphology of KGN cells and significantly stimulated KGN cell migration. The morphological transition and enhanced migratory ability of TGFα-treated KGN cells suggests that TGFα may facilitate or promote GCT cell metastasis.
Although EGFR signaling pathways in ovarian epithelial cancer cells have been described 
, very little has been shown regarding the signaling pathways activated by TGFα and EGF in GCTs. Results
in the present study suggest that TGFα rapidly activates the PI3K/Akt/mTOR and Raf/MEK/ERK1/2 signaling pathways. Our findings are partially consistent with Zhang et al., who recently studied the role of HOXA7 in the regulation of granulosa cell proliferation and showed that EGF transiently stimulated phosphorylation of ERK1/2 and Akt in KGN cells 
. Our results showed that while TGFα stimulated a transient activation of Akt, it stimulated a sustained activation of ERK1/2; in fact TGFα-stimulated activation of ERK1/2 was sustained for more than 3 days (). The duration of ERK activity has been implicated as a critical factor in cell fate decisions 
. Whereas transient ERK activation causes cell proliferation, the overexpression of EGFR induced a sustained ERK activation leading to differentiation of PC12 neuronal cells 
. In contrast, in CCL39 Chinese hamster lung fibroblasts it is sustained, but not transient, activation of ERK that is required for the proliferation of quiescent fibroblasts 
. Immediate early genes downstream of ERK activation, such as Fos, Jun, Myc and Egr1, encode critical transcription factors for cell proliferation. Furthermore, sustained ERK activation causes phosphorylation and stabilization of the proteins encoded by these genes. For example, cyclin D1, which is important for S-phase entry and under the regulation of AP-1 (Jun and Fos proteins), is elevated and maintained by sustained ERK activation 
. The prolonged activation of ERK by TGFα in GCT cells suggests that TGFα might be a major regulator of GCT cell fate. However, treatment with U0126, which effectively inhibited ERK activation in response to TGFα, was unable to completely suppress the proliferation of KGN cells. The function of TGFα-induced sustained activation of ERK in GCT warrants further investigation.
Cyclin D2 and p27Kip1, which function in the G1-S phase transition, are critical cell cycle regulators in ovarian granulosa cells 
. In the present study, treatment of KGN cells with TGFα hours significantly increased cyclin D2 expression and suppressed p27Kip1 expression. The changes in cyclin D2 and p27Kip1 may explain the increased number of KGN cells in the S and G2/M phases after TGFα treatment. The present in vitro findings with human GCT cells are supported by studies using mutant mouse models. Cyclin D2 deficient females mice are sterile because the granulosa cells are unable to proliferate normally in response to FSH 
. Tumorigenesis in the α-inhibin deficient mice is accompanied by an increased expression of cyclin D2 and a decreased expression of p27Kip1 
. Mutant mice with double knockout of p27Kip1 and α-inhibin developed and succumbed to ovarian tumors more rapidly than α-inhibin knockout mice. However, cyclin D2 and α-inhibin double-knockout mice lived longer than mice lacking α-inhibin alone 
, suggesting that p27Kip1 acts cooperatively with inhibins to negatively regulate granulosa cell proliferation 
and cyclin D2 may antagonize the tumor-suppressing actions of p27Kip1. Taken together, these findings suggest that TGFα may be involved in granulosa cell tumor initiation and progression. More experiments are required to reach a final conclusion.
The components of PI3K/AKT/mTOR signaling pathway are frequently overexpressed in human epithelial ovarian cancer and are attractive targets for epithelial ovarian cancer therapy. Phase I–II trials are currently evaluating mTOR inhibitors in ovarian cancer patients 
. However, little is known about the expression and action of components of this pathway in GCT. One recent study examining 12 GCTs showed that mTOR was expressed in every tumor sample and phosphorylated mTOR was detected in 30% of the tumor tissues 
. This study correlated the presence of mTOR with the expression of HIF1α and VEGF, both of which are important for tumor angiogenesis. In the present study, we demonstrated that TGFα, via EGFR, stimulated phosphorylation of mTOR and its downstream target proteins p70S6K and rpS6. This pathway appears to be critical for the actions of TGFα on KGN cell proliferation as our studies demonstrate that proliferation was disrupted by treatment with inhibitors of PI3K and mTOR. As expected, the activation of Akt and mTOR signaling in response to TGFα was rapid and the PI3K inhibitor blocked both AKT and mTOR signaling. However, Akt signaling was transient and returned to base line after 30 minutes while the phosphorylation of the mTOR targets p70S6K and rpS6 continued to increase over the 2 hour time course. This suggests that TGFα can maintain mTOR signaling independent of ongoing Akt signaling in KGN cells. In support of this idea, Fan et al. reported that in glioma cells the EGFR can activate mTOR independent of Akt by a mechanism involving protein kinase C 
. While the cellular mechanism responsible for the sustained elevation in mTOR signaling in KGN cells awaits investigation, our study clearly demonstrates that the mTOR inhibitor rapamycin effectively suppressed TGFα-induced proliferation of KGN cells. Thus, mTOR-targeted therapy may represent a strategy for reducing vascularization of GCT 
and inhibiting growth of GCTs that express TGFα and ErbB receptors. Furthermore, dual PI3K and mTOR inhibitors may provide additional therapeutic benefit 
In summary, our present study suggests that TGFα, via ErbB receptors, promotes KGN GCT cell cycle progression, enhances tumor cell proliferation and facilitates GCT cell migration. Moreover, human GCT cells express the ligand TGFα and its ErbB receptors suggesting that components are available in human GCTs to promote the proposed actions of TGFα on GCT cell proliferation and facilitate tumor cell migration in an autocrine and/or paracrine manner. Our results also indicate that multiple signaling pathways are involved in TGFα regulation of GCT cell growth and migration.