The Role Of Estrogen Receptors In The Invasiveness Of Nonfunctional Pituitary Adenomas

Pituitary adenomas (PAs) comprise 15 to 20% of primary intracranial tumors. Though they are benign tumors, PAs often invade surrounding structures such as sphenoid sinus and cavernous sinus. Total surgical removal of invasive adenomas remains a challenge for surgeon, for the local invasion of the tumor mass. Due to re-growth of residual tumor, invading PAs are associated with higher recurrence rate and they are considered as an intermediate stage in tumor progression towards carcinomas. Clarification of the factors affecting tumorous invasion is essential for evaluation of prognosis in patients and developing new adjuvant treatment.Estrogen, through ERs (ERαand ERβ), affects cellular proliferation and hormone synthesis in pituitary gland. Recent studies have demonstrated that there is a correlation between ER level and tumor growth. In GH3 cell line, ERαis able to up-regulate the transcription of key growth-promoting genes. Moreover, in female ERβ–/–mice loss of ERβlead to development of large gonadotropin-positive pituitary tumors when the mice are two years of age. As similar as double knockout ERαβ–/– mice, ERα–/– and male ERβ–/–mice do not develop pituitary tumors. These results indicate that an imbalance between ERαand ERβcontributes to pathogenesis and biological behavior of PAs; however, the underlying mechanism is unclear.Disconnection and detachment of cells are crucial for tumor invasion. Loss or down-regulated of E-cadherin may contribute to the development of tumor invasion in a wide variety of human malignancies. In prolactinomas, loss of membranous staining of E-cadherin was frequently associated with tumor size and local invasion. These results imply that E-cadherin level may contribute to invasiveness of PAs. Several other series of studies showed that E-cadherin level was affected by estrogen receptor activation, but no studies have investigated the relationship between E-cadherin and estrogen receptors in pituitary tumors. Slug is a member of the snail family of transcription factors; it repressed the expression of E-cadherin. Both in vitro and in vivo studies have suggested that the expression of E-cadherin is inversely correlated with the expression of slug in tumorigenesis. Although the relationship between slug and E-cadherin are being elucidated in many tumors of epithelial origin, comparatively much is unknown about slug regulation in PAs.The nonfunctional pituitary adenoma (NFPA) is one of the most common PAs. In contrast to other PAs characterized by hormonal excess, NFPAs are characterized by null cells or endocrine-inactive neoplasms of anterior pituitary. In this study, we studied the expressions of ERs in invasive and noninvasive NFPAs tissues using immunohistochemistry and evaluated whether these parameters are associated with tumor invasiveness. Expressions of E-cadherin and Slug, PCNA-labeling index in invasive and noninvasive NFPAs were evaluated as indicators of tumor invasiveness. In addition, the roles of ERαand ERβagonist (PPT and DPN respectively) in the migration and proliferation of primary cultured pituitary adenoma were investigated.The study consists of two parts:Part 1: The expression of ERs in the NFPAsNFPAs tissues were obtained from 41 patients at Xin Qiao Hospital and the patients all had transsphenoidal surgery via a sublabial or transnasal approach. Epidemiological analysis was performed to study the correlation between estrogen level in the serum and invasiveness of NFPAs. The expression of ERαand ERβin the NFPAs were detected by immunohistochemistry, the correlation between invasion and proliferation in NFPAs was evaluated with PCNA and Ki-67 labeling index. In addition, the expression of E-cadherin and its repressor Slug were analyzed to understand the potential mechanism that ERs affect the invasion of NFPAs.Results:1. The occurrence of invasive type was statistically significantly lower among men compared with woman. There is no significant difference in the serum E2 concentrations of patients with invasive NFPAs between females and males, whereas nuclear ERαstaining in the invasive NFPAs was significantly stronger in the female patients than that from male patients. 2. ERαexpression was deteced in 37 of 41 (90%) samples and in all 15 invasive adenomas. For ERβ, nuclear staining in noninvasive adenomas was positive in 25 of 26 (96%) samples, whereas in invasive ones, expression was lost in 9 of 15 (60%) samples. Moreover, compared with noninvasive NFPAs, expression of ERαwas extremely higher in the invasive ones, whereas the expression of ERβwas significantly lower. These results indicate that ERs may play an important role in the regulation of NFPAs invasiveness, in which ERβprobably inhibits this process whereas ERαcontributes to it.3. Strong expression of PCNA was detectable in all 41 samples; however, the PCNA labeling index was not different between invasive NFPAs and noninvasive ones. Similar to expression of PCNA, there is no significant difference in the Ki-67 labeling index between these two groups. Then we infer that proliferation was not always associated with invasion of PAs,nor associated with the expression of ERαand ERβ.4. The levels of E-cadherin and Slug were detected using semi-quantitative RT-PCR. Low expression of E-cadherin was detected in five invasive NFPAs, whereas expression of Slug in the noninvasive NFPAs was high. Slug mRNA was upregulated in the invasive NFPAs compared with noninvasive ones. These differences were confirmed at the protein level by Western blotting and immunohistochemistry. Furthermore, level of Slug was negatively correlated with ERβand positively correlated with ERα; whereas level of E-cadherin was negatively correlated with ERαand positively correlated with ERβ. It seemed that ERs may act on the Slug-E-cadherin pathway to affect invasion of NFPA.Part 2: The effects of ERs agonist on primary culture cells of invasive NFPAsERs are ligand-activated nuclear receptors; their natural ligand is 17β-Estradiol (E2). Two synthetic compounds of PPT and DPN have been identified as special agonists for ERαand ERβrespectively. In this study, endogenous ERs in the primary cultured cells were activated by E2, PPT and DPN administration into culture medium. The proliferation of cells was evaluated by MTT assay and the invasion of the cells was detected by transwell invasion assay. Furthermore, both mRNA and protein levels of E-cadherin and Slug were detected in vehicle and agonist treated groups to analyze the relationship between ERs and E-cadherin or Slug.1. Both ERαand ERβwere expressed in the primary cultured cells derived from invasive NFPAs, of which ERαwas the predominate ER with high expression level. Colocalization of ERαand ERβwas observed with double immunofluorescence, of few double stained cells were detected under microscope.2. There is no significant difference in the cellular proliferation of primary cultured cells derived from invasive NFPAs among agonist treated group and control group. This may infer that ERs activation had no significant effect on NFPA cell proliferation in vitro.3. The invasion of cells was evaluated by transwell invasion assay. We found that cells treated with either ERαagonist PPT or E2 exhibited high invasion, whereas the cells treated with DPN exhibited repressed invasion.4. After treated with ERαagonists PPT or E2, the cells showed upregulated Slug expression and downregulated E-cadherin expression compared with cells treated with vehicle. Conversely, ERβactivation with DPN in the cells induced downregulated Slug expression, and upregulated E-cadherin expression.Conclusions1. In vivo, expression of ERαwas extremely higher in the invasive NFPA compared with noninvasive ones, whereas the expression of ERβwas significantly lower. In vitro, the activation of ERαinduced high invasion of the tumor cells, otherwise, the activation of ERβrepressed the invasiveness. These results indicate that ERs may play an important role in the regulation of NFPAs invasiveness.2. In NFPA tissues, decreased expression of E-cadherin was associated with tumor invasion, and expression of E-cadherin was downregulated by its repressor Slug. Moreover, this was confirmed by in vitro study.3. Both in vivo and vitro, the result showed that ERβactivation induced downregulated Slug expression, and upregulated E-cadherin expression which repressed NFPAs invasiveness, otherwise, ERαactivation act in the opposite direction.In conclusion, the ERαand ERβmay be involved in the invasiveness of NFPA with opposite way, of which ERβinhibits this process whereas ERαcontributes to it. The underlying mechanism is further to be demonstrated to be associated with the Slug-E-cadherin pathway.