Abstract 2632: SPARC modulates melanoma cell size, adherence, actin architecture and migration through Akt/S6K and Rac1 regulation.
Authors: Salvatierra Edgardo , Leishman Claudia , Alvarez Mariano , Podhajcer Osvaldo 
Published: August 2014

Abstract
Cancer cells display intermediate levels of adherence and a poorly organized actin cytoskeleton. These characteristics provide a favorable state for initiating dynamic processes -such as cell migration- that ultimately lead to invasion of the neighbor stroma and metastatic foci implantation.During the last years, it has become evident that the extracellular matrix (ECM) is the source of several molecules that regulate these processes, being the matricellular family of proteins one of the most important ones. SPARC (Secreted Protein Acidic and Rich in Cysteine) is a matricellular protein whose overexpression in malignant or stromal cells was often associated with increased aggressiveness and bad prognosis in melanoma as well as in a wide range of other human cancers. In previous studies, we and others have reported that SPARC plays a key role in melanoma progression including the modulation of cell capacity to migrate. Here, we describe the underlying molecular mechanism through which SPARC modulates this process.SPARC knockdown (KD) in A375 and IIB-MEL-LES melanoma cells induced dramatic changes in cells’ size and actin architecture. SPARC-KD cells showed an enlarged size and an ordered actin CSK with actin bundles spanning all the cell body and ending with prominent focal contacts at the edge of the cell. These cells also showed lamellipodia formation and accelerated spreading in serum. On the other hand, SPARC-KD melanoma cells evidenced strongly restricted capacity to spread on type IV collagen and laminin that was associated with downregulation of α6 integrin and upregulation of β1 integrin. Both the re-expression of SPARC as well as the addition of the protein were able to reverse the cellular phenotype and integrin levels.Small GTPases are intermediate factors that regulate lamellipodia formation, cell spreading and integrins activity. Transient transfection of dominant negative (DN) or constitutively active sGTPases pointed to Rac1 as being involved in this process. G-ELISA activation assays confirmed increased Rac1 activity in SPARC-KD cells. The diminished capacity of SPARC-KD cells to migrate was restored by SPARC re-expression, exogenous addition and by expression of Rac1-DN. Interestingly Rac1-DN, expression also restored α6 and β1 integrin.Further studies on mediators of SPARC's effect demonstrated an increased phosphorylation of cSrc and S6K with inhibition of AKT phosphorylation in cells SPARC-KD. Remarkably, the exogenous addition of SPARC to SPARC-KD cells increased AKT phosphorylation and decreased S6K phosphorylation concomitantly with an augmented cell migration.Taken together, these results show that SPARC promotes cell migration through the modulation of an intracellular pathway that involves cSrc/AKT/S6K and Rac1 that affects cell shape, including cell size, adherence and actin architecture.
Citation Format: Edgardo Salvatierra, Claudia Leishman, Mariano Alvarez, Osvaldo Podhajcer. SPARC modulates melanoma cell size, adherence, actin architecture and migration through Akt/S6K and Rac1 regulation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2632. doi:10.1158/1538-7445.AM2013-2632