TGF-β signaling is disrupted in endometrioid-type endometrial carcinomas

Abstract

Objective

Previous studies have demonstrated deregulation of the expression and changes in the intracellular distribution of TGF-β pathway components in human endometrial cancer (EC). The aim of this study was to assess the relationship between the expression of TGF-β cascade components, including TGF-β receptor type I (TGFβRI) and type II (TGFβRII), SMAD2, SMAD3, SMAD4, and clinicopathological features—tumor grade, FIGO classification, and depth of myometrial invasion—of type I (endometrioid-type) ECs to give some insight into the role of TGF-β cascade components in endometrial tumorigenesis.

Methods

The expression of TGFβRI, TGFβRII, SMAD2, SMAD3, and SMAD4 was evaluated both at the mRNA and protein level using reverse transcription polymerase chain reaction (RT-PCR) and ELISA, respectively.

Results

Infiltrating endometrial carcinomas (less and more than half of the myometrial wall thickness) express significantly higher TGFβRII protein level compared with non-infiltrating tumors (P = 0.04 and P = 0.01, respectively). Decreased level of SMAD2 and SMAD4 mRNAs was observed in the uterine tumors infiltrating less and more than half of the myometrial wall (P = 0.03 and P = 0.02, respectively) compared with noninfiltrating ECs. Significantly higher SMAD4 protein level in the cytoplasmic fraction of ECs was found when tumor grade and depth of myometrial invasion were considered (P < 0.05). Generally, tumor progression was associated with a decreased number of cases characterized by the presence of SMADs in the nuclear fraction only.

Conclusion

Our data suggest that disturbances of the TGFβRII and SMAD4 expression as well as localization of SMADs may be important to the infiltration of the myometrial wall by the type I endometrial carcinomas.

Introduction

Transforming growth factor β (TGF-β) is a member of the large family of structurally related cytokines that play an important role in controlling cell proliferation, differentiation, migration, and apoptosis [1], [2]. TGF-β signaling is initiated by ligand binding to specific transmembrane serine/threonine kinase TGF-β receptors such as type II (TGFβRII), which in turn transphosphorylates type I receptor (TGFβRI). Receptors convey signals across the plasma membrane through intracellular effectors such as SMAD proteins, which are translocated to the nucleus where they act as transcription factors [3], [4], [5], [6]. SMAD proteins have been divided into three functional classes: receptor-regulated (R-SMADs), common mediator (Co-SMAD), and inhibitory (I-SMADs) [5], [6]. Representatives of the first class are directly phosphorylated by TGFβRI and are involved in the various signaling pathways. SMAD2 and SMAD3 are effectors of TGF-β/activin signaling, while SMAD1, SMAD5, and SMAD8 are involved in BMP (bone morphogenetic protein) signaling. Common mediator SMAD4 is critical for TGF-β/activin and BMP signaling cascades. Representatives of the third class of SMADs (SMAD6 and SMAD7) have the ability to inhibit the TGF-β signaling pathway [7], [8].

TGF-β signaling through SMADs may integrate information from distinct signaling pathways, thus introducing the possibility that SMADs play important roles in the intracellular network and in the complexity of the responses to TGF-β and related growth factors [9], [10]. Although regulation of the TGF-β signaling pathway has been extensively studied, many aspects are still unclear and should be further elucidated. TGF-β can play multiple roles in human tumorigenesis, behaving as a tumor suppressor at early stages and a tumor promoter at late stages of carcinogenesis [11], [12], [13]. It has been well documented that deregulation and/or inhibition of the TGF-β signaling through SMADs may be very important for neoplastic transformation and as well as tumor progression [2], [12], [13].

Our laboratory is focused on determining the role of the TGF-β signaling cascade in the development and progression of endometrial cancer. In our first study, although no significant differences in the TGF-β receptor mRNA level between normal and carcinomatous endometrium was observed, we did see significantly lower TGFβRI (P = 0.028) and significantly higher TGFβRII (P = 0.007) protein levels in cancerous endometrium compared with normal endometrial tissue [14]. Moreover, differences in SMAD protein expression and intracellular localization between neoplastic and normal endometrium were noted [15]. To expand this research, this study sought to analyze the relationship, if it exists, between changes in the TGF-β signaling cascade and clinicopathological features of cancer of the uterine corpus. The expression of TGF-β receptor type I and type II as well as SMAD2, SMAD3, and SMAD4 was analyzed both at the mRNA and protein level in relation to the patient's age, tumor grade, FIGO classification, and depth of myometrial invasion using RT-PCR and ELISA techniques, respectively. Simultaneously, intracellular distribution of SMAD with regard to the clinicopathological features of endometrial cancers was examined.