Experimental Study On The Role Of ADAM10 In Regulation Of Carvernous Sinus Invasion In Pituitary Adenomas

BackgroundPituitary adenomas are common lesions believed to account for 10% to 15% of all primary brain tumors. Epidemiologic estimates indicate an annual incidence of 8.2 to 14.7 cases per 100,000 people. It usually lead to severly physical and mental health problems. There is clear difference in the pathogenesis and biological behavior between pituitary adenomas and cancer in the general sense, the tumor size and invasion are not entirely a reflection of cell proliferation and tumoral growth rate. Researches commonly used in tumor include cell proliferation, cell cycle, cell death and differentiation, are not really from the perspective of pituitary tumors to illustrate and solve the problem. Study of tumor invasion and metastasis, appropriately becomes focus of current pituitary tumor research. A large number of literature study abandoned the tumorigenicity, and shifted their research priorities to the mechanism of invasive pituitary adenomas. This is exactly the difficult and key issues in clinical treatment of pituitary adenomas. Currently, the diagnosis of invasive pituitary still relies on imaging, histopathology, and direct observation during surgery, but the use of histopathological dural invasion is restricted by the difficulties in getting dura specimens, while the imaging and intraoperative observation usually exist subjective factors, thus the results often vary and the repeatability, accuracy is limited. The objective molecular markers should have sufficient sensitivity and could judge the invasive potential of tumor in a early stage. They should be promising indicators to guide clinicians modify their surgery or drug treatment strategy. Further studies may develop specific inhibitors according to the invasive molecule, and provide the foundation for future gene therapy.ADAMs (A disintegrin and metalloproteinases) are a new gene family, containing a disintegrin domain and sharing the metalloproteinase domain with matrix metalloproteinases (MMPs). They are structurally classified into two groups: the membrane-anchored ADAM and ADAM with thrombospondin motifs (ADAM-TS). These molecules are involved in various biological events such as cell adhesion, cell fusion, cell migration, membrane protein shedding and proteolysis. Studies on the biochemical characteristics and biological functions of ADAMs are in progress, and accumulated lines of evidence have shown that some ADAMs are expressed in malignant tumors and participate in the pathology of cancers. ADAM10 is a member of the ADAMs and is highly expressed in many tumors. It can process several critical signaling molecules associated with cancer development and tumor progression including ErbB, Notch and several adhesion molecules. The prototypical ADAM protein has a prodomain, metalloproteinase domain, disintegrin domain, cysteine–rich EGF-like domain, and transmembrane and cytoplasmic domains.ADAM10 protein anchorage in the cell membrane after activation and proteolytic cleavage of various substrates of inflammation and cancer, Involving in the pathogenesis of inflammation and cancer. Its substrates include the adhesion molecule L1 and CD44. CD44 is a typeâ… transm em brane cell surface adhesion m olecules involved in tum or invasion and metastasis. It is expressed in most human cells. The high level of CD44 protein is also found in many types of cancer. CD44 proteins gain their function depending on the ectodomain cleavage, which is regulated by ADAM10. The ectodomain cleavage can produce large amounts of soluble CD44, subsequently leading to decreased cell adhesion. This process may prompt the tumor cells isolate from the extracellular matrix, which is an important step in tumor cell migration. L1 adhesion molecule is another known ADAM10 substrate L1 is a type I transmembrane glycoprotein of the immunoglobulin family that has important functions for the development of the nervous system by regulating cell adhesion and migration. L1 overexpression was found in a variety of tumors, and was correlated with enhanced tumor invasiveness and metastasis.We first used immunohistochemistry, real-time quantitative PCR and Western blot technology to verify the mRNA and protein level of ADAM10 in gross specimens and GH3 cell line, and analyzed its relationship with tumor invasiveness. Then we studied the gene function of ADAM10 in GH3 cell line, using genetic engineering technology such as RNA interference and overexpression. The aim of this study is to clarify the exact role and mechanisms of ADAM10 in the cavernous sinus invasion in pituitary adenomas and to find invasive molecular markers, fortunately, which could provide guidance for clinical decision making and open up a new direction for gene therapy.Partâ… :Expression of ADAM10 in pituitary adenomas and its significanceObjective: To investigate the expression level of ADAM10 and its substrate CD44, L1 In invasive adenomas and non-invasive adenomas; To study the CaM-ProADAM10, Src-Shc interaction differences between the two groups; and explore their correlation with the clinical data.Methods: All adenoma samples were divided into invasion group and non-invasion group according to Knosp grading criteria and divided into functional adenomas group and non-functional adenomas group based on the clinical manifestation and results of endocrine secreting level. The ADAM10 transcription level was detected using quantitative real-time PCR. Cellular localization and protein level of ADAM10, Src, ERK were explored with immunohistochemisty. The protein expressions of ADAM10, L1, CD44, E-cadhein, Vimentin in these samples were detected using western blotting. The CaM-ProADAM10 or Src-Shc interaction was studied using co-Immunoprecipitation.Results: 1. Both the mRNA level and protein level of ADAM10 were higher in invasive adenomas group than non-invasive group. The ADAM10 protein immunoreactivity located in cellular membrane of pituitary adenoma cells. 2. The staining differences of ADAM10 was not associated with age, gender and secreting status. Both p-Src and p-ERK immunoreactivity were closely related to ADAM10 expression respectively, and both have statistical significances(p=0.036, 0.035 respectively). 3. The ectodomain cleavage levels of substrate molecules, CD44 and L1, were higher in invasive group(p<0.05). 4. The co-Immunoprecipitation showed CaM-ProADAM10 interaction weakened(p<0.05), while the Src-Shc interaction enhanced(p<0.05) in invasive pituitary adenomas group. 5. Migration-related molecular marker, E-cadhein was expressed higher in the invasive group(p<0.05), and Vimentin showed an opposite trend(p<0.05).Conclusions: 1. Overexpression of ADAM10 may serve as an important molecular mechanism that underlies the development and progression of cavernous sinus invasion. 2. Role of ADAM10 protein in enhancing tumor cell invasiveness possibly through cleavage adhesion molecules L1 and CD44 and further increases cell migration. 3. ADAM10 activation in invasive pituitary adenomas may be induced by the weakened CaM-ProADAM10 interaction. 4. The substrate cleavage features of ADAM10 may be associated with strenthened Src–Shc interaction and further activation of ERK pathway.Partâ…¡:Expression of ADAM10 in GH3 cell lineObjective: To investigate the expression of ADAM10 in GH3 cell line; the ADAM10 activation diffenences under increased Ca²⁺ influx or blocked Ca²⁺ influx using EGTA circumstances; the CaM-ProADAM10 interaction differences and cleavage changes of CD44 when Ca²⁺ influx was increased; the changes of L1 cleavage, Src phosphorylation, and Src-Shc interaction when treated GH3 with PMA. Finally, Laser Scanning Confocal Microscope was used to directly observe the impact of Ca²⁺ influx on CD44 cleavage and PMA treatment on L1 cleavage.Methods: Mechanical Scraping technology and adding ionomycin method were separately used to increase the Ca²⁺ influx. EGTA was used to block Ca²⁺ influx, and cells were collected at 0min, 15min, 30min for testing. Co-Immunoprecipitation was used to determine CaM-ProADAM10 interaction changes after Mechnical Scraping treatment, and the Src-Shc interaction changes after PMA treatment. Western blotting was used to detect cleavage of CD44 after Ca²⁺ influx, and cleavage of L1 after PMA treatment. Laser Scanning Confocal Microscope was used to directly observe the impact of Ca²⁺ influx on CD44 cleavage and PMA treatment on L1 cleavage.Results: As the Ca²⁺ influx increased, the activation of ADAM10 and cleavage of CD44 increased, while the CaM-ProADAM10 interaction weakened. The opposite results appeared when Ca²⁺ influx was blocked. The cleavage of L1 and Src Phosphorylation increased after PMA treatment, also the Src-Shc interaction enhanced. The confocal results showed both L1 and CD44 co-localized with ADAM10 in the cell membrane. It could be directly seen that CD44 cleavage increased after Ca²⁺ influx and L1 cleavage increased after PMA treatment.Conclusions: 1. There exist ADAM10 protein expression and L1 or CD44 cleavage in GH3 cell line at low levels. 2. Ca²⁺ influx could reduce the inhibitory effect of CaM on ADAM10, since this the ADAM10 protein activated and futher cleavage the CD44 molecules. 3. PMA treatment could enhance the Src-Shc interaction, next activate the ERK pathway, and futher enhance the cleavage of L1 molecules.Partâ…¢: Over-expression or RNA interference targeting ADAM10 can change the biological behavior of GH3 cells in vitroObjective: To study the gene function of ADAM10, using RNA interference and gene overexpression techniques.Methods: 1. In RNA interference group: Western blot was used to detect L1 and CD44 cleavage level and Src phosphorylation level and L1 cleavage level after blocking ERK pathway using its inhibitor PD; Laser Scanning Confocal Microscope was used to directly observe the impact of RNA interference on CD44 and L1 cleavage. 2. In gene over-expression group using Lentivirus method: Western blot was used to detect L1 cleavage and Src phosphorylation level; Laser Scanning Confocal Microscope was used to directly observe the impact of gene over-expression on E-cadherin and Vimentin expression.3. Using cell detachment assay to explore the cell adhesion changes after RNA interference; Using transwell migration assay to explore the cell invasion changes after RNA interference.Results: In GH3 cell line, the cleavage level of L1 and CD44 decreased obviously after the endogenous ADAM10 was interferenced by ShRNA, furthermore, the cleavage of L1 would further decrease when the ERK inhibitor PD98059 was introduced. The cleavage of L1 and phosphorylation of Src were enhanced after over-expression ADAM10. The confocal results showed both cleavage of L1 and CD44 were decreased after ShRNA, expression of E-cadherin increased and Vimentin decreased after over-expression. The cell detachment assay showed cell adhesion ability enhanced while the suspension cell decreased. ADAM10 shows great influence on cell migration on Hyalluron, in transwell experiments.Conclusion: 1. The cleavage of L1 and CD44 decreased when interferenced the endogenous ADAM10, but the cleavage level enhanced when over-expression ADAM10, these facts indicates that ADAM10 play an key role in cleavaging of CD44 and L1. 2. Src-ERK pathway participated in the cleavage of L1. 3. The results that GH3 cell adhesion ability increased and migration ability decreased after ShRNA confirmed that ADAM10 play an important role in modulating cell adhesion and migration.