Functional characterization of ADAM23 gene splicing isoforms

Author(s)

Felicia Peterson Cavalher

• Document type: Doctoral Thesis
• Press: São Paulo.
• Institution: Universidade de São Paulo (USP). Conjunto das Químicas (IQ e FCF) (CQ/DBDCQ)
• Defense date: 2012-09-13

Examining board members

Anamaria Aranha Camargo; Roger Chammas; Bettina Malnic; Eduardo Moraes Rego Reis; João Paulo de Biaso Viola

• Advisor: Anamaria Aranha Camargo; Erico Tosoni Costa

Abstract

ADAM23 is a transmembrane glycoprotein that belongs to the ADAM (A Disintegrin and Metalloprotease) family of proteins and exhibits the typical protein structure of the family members, but it doesn\'t have metalloprotease activity. The ADAM23 gene has three splicing isoforms, α, β and γ, that code for proteins with different C-terminal regions. Isoforms α and β code for proteins with different transmembrane domains, while γ probably constitute a secreted or cytoplasmatic isoform of ADAM23. It has been demonstrated that the ADAM23 gene is epigenetically silenced in advanced stage breast tumors and that its silencing is associated with a higher risk of developing metastases and with a worse prognosis. Recently, it was described that ADAM23 protein interacts directly with αVβ3 integrin in the breast tumor cell line MDA-MB-435, modulating its conformational state and controlling its activation. Using RNAi, it was observed that the complete silencing of ADAM23 gene (the three isoforms) raises the levels of αVβ3 in its active conformation in the surface of MDA-MB-435 cells, promoting an increase in its migratory and adhesive capacity. In the present work, we evaluated by real time PCR the expression pattern of the three splicing isoforms of ADAM23 gene in five normal tissues (breast, colon, brain, prostate and pancreas) and in twelve tumor cell lines derived from these tissues. We observed differences in the expression levels of the three isoforms in all samples, either within a specific sample or comparing normal tissues among them or with tumor cell lines. Isoform γ has the highest expression in all normal tissues (except for brain) and in all tumor cell lines evaluated. In breast and prostate normal tissues and in all tumor cell lines, ADAM23α is the second most expressed isoform, while β is the less expressed. We also noticed that the ratio represented by each isoform, relative to the total expression of ADAM23 gene, is altered in the tumor cell lines, compared to the corresponding normal tissues. With the aim to elucidate the function of ADAM23 isoforms separately, we used shRNAs (short hairpin RNAs) to reduce the expression of each isoform specifically in the MDA-MB-435 tumor cell line, and studied its effects in proliferation, morphology, adhesion and cell spreading. We observed that the reduced expression of isoform γ significantly increased the proliferation rate of MDA-MB-435 cells cultivated in tridimensional system. Also, we demonstrated that ADAM23γ participates in the regulation of cell morphology and spreading of MDA-MB-435 cells, both in standard culture conditions (cell culture media with fetal serum and in plates not sensitized with substrates) and in specific components of extracellular matrix, such as fibronectin, collagen type I and matrigel. Isoform α is also involved in the control of morphology and spreading of MDA-MB-435 cell line, although in a distinct manner from isoform γ. ADAM23β doesn\'t interfere in the morphology of MDA-MB-435 cells and plays a discrete role in cell spreading only under standard culture conditions. Together, our results demonstrate that ADAM23 isoforms are differently expressed in normal and tumoral tissue, and play distinct biological roles. (AU)