Most eukaryotic cells sense motogenic signal gradients in their microenvironments and respond through cell polarization  and expand a single lamellipodium to establish directional migration . Switching from the stationary state of the cell to the mobile state as in wound healing, gastrulation or metastasis depends on the actin cytoskeleton .
Migration and invasiveness of cancer cells is the hallmark of malignancy . Cell migration is a highly integrated multistep process that includes development of cytoplasmic protrusions, attachment and traction . The formation of these protrusions is driven by spatial and transient regulation of actin polymerization at the leading edge of polarized migratory cells . Actin filament (F-actin) dynamics are regulated by actin binding proteins (ABPs) which are responsible for polymerization and treadmilling . One of the most important families of ABPs is the ADF/cofilin (AC) family of proteins .
Vertebrates express three isoforms of ADF/cofilin encoded by three different genes: Actin Depolymerizing Factor (ADF), also known as destrin in mammals, non-muscle cofilin-1 (Cfl-1), and cofilin-2 (Cfl-2), which is enriched in muscle cells [8–10]. Human ADF and cofilin-1 are more than 70% identical in amino acid sequence . At low concentrations with respect to actin subunits, ADF and cofilin-1 sever the filaments, but at higher concentrations they bind cooperatively to saturate F-actin and stabilize the severed fragments [9, 12]. Also, ADF/cofilin depolymerize F-actin from the pointed end leading to enhancement of treadmilling . ADF-actin has a much higher critical concentration for assembly than does cofilin-actin [11, 14], and thus ADF but not cofilin can serve as a major monomer sequestering protein [15, 16].
Metazoan ADF/cofilins are regulated by phosphorylation/dephosphorylation of a conserved serine (encoded ser 3 in human proteins) . Known kinases are LIM Kinases LIMK1, LIMK2, and Testicular Kinases TESK1 and TESK2 . The more specific cofilin phosphatases are chronophin and slingshot (SSH) [18–20]. AC proteins are pH-dependent in their interactions with F-actin [16, 21–24].
Most research on ADF/cofilin proteins in metastatic invasion has focused on cofilin-1 (hereafter referred to as cofilin). Although ADF and cofilin can substitute for one another for many housekeeping activities in cultured cells , this is not the case during development. Cofilin null mice are not viable despite the fact that ADF is upregulated . In contrast, ADF null mice are viable but show abnormal corneal thickening, suggesting that cofilin can rescue the lack of ADF except in corneal epithelial cells . However, in ureteric bud (UB) epithelium, ADF and cofilin show considerable functional overlap, whereas simultaneous lack of both genes arrested branching morphogenesis at an early stage . Likewise, most forms of ADF and cofilin from across phylogeny are able to compete similarly with myosin II for F-actin binding .
Silencing cofilin in colorectal cancer cells (Isreco1) did not interfere with their ability to undergo transwell migration across collagen in response to a chemotactic attractant. On the other hand, silencing of ADF, which represented only 17% of the total ADF/cofilin, significantly inhibited transwell migration, strongly suggesting different cellular functions of each protein in these cells .
Several studies have demonstrated an increase in cofilin amounts or in activity (dephosphorylated form) in cancer cells including cell lines derived from T-cell lymphoma (Jurkat) and carcinomas from the cervix (HeLa), colon (KM12), liver (HepG2) and kidney (COS1) , and in clinical tumor samples of oral squamous-cell carcinoma , renal cell carcinoma  and ovarian cancer . In addition, overexpression of cofilin increases velocity of cell migration in Dictyostelium  and human glioblastoma cells . Expression of wildtype or a non-phosphorylatable cofilin mutant in which ser 3 has been mutated to alanine (S3A) increases melanoma cell invasion .
However, opposite findings have also been reported. LIMK 1 activity, which should decrease active cofilin, is upregulated in invasive breast and prostate cancer cell lines and its overexpression increased motility of tumor cell lines [37, 38]. Furthermore, suppression of LIMK2 in human fibrosarcoma cells or expression of a dominant negative LIMK1 in an animal model of tumor invasion, limited cell migration and efficiency to form dense colonies without affecting cell proliferation rate or viability [37, 39, 40]. Such opposite findings suggest that targets of LIMK1 and LIMK2, which include ADF as well as cofilin , bring about different effects, which could be dependent on relative amounts of ADF or cofilin that are expressed in the different tumor cell types.
MTLn3 mammary adenocarcinoma cells have been used extensively in the study of metastasis. In breast tumor microenvironments, gradients of EGF secreted by tumor associated macrophages (TAMs) act as chemo-attractants leading to cancer cell polarization toward EGF . EGF binds to EGF receptor (EGFR) on the surface of MTLn3 cells leading to the activation of phospholipase Cγ (PLC-γ) and phosphatidylinositol 3-kinases (PI3K). ADF/cofilin are bound to phosphatidylinositol 4,5-bisphosphate (PIP2) in the plasma membrane of resting MTLn3 cells . EGF-activated PLC-γ hydrolyzes PIP2 causing the release of ADF/cofilin from plasma membrane . Active ADF/cofilin severs actin filaments creating new barbed ends that serve as nuclei for polymerization. New ATP-actin or ADP-Pi-actin subunits are preferred by the Arp2/3 complex, which is responsible for creating the branched actin filament arrays at the leading edge of migrating cells forming cell protrusions needed for crawling [44–47].
To study the roles of ADF and cofilin in cancer cell migration, we selected MTLn3 cells that expresses nearly identical amounts of each protein and silenced each in turn while performing a number of assays to assess the role of each in different aspects of polarized migration. Our results suggest that whereas many of the functions of cofilin and ADF are redundant, each of these isoforms has subtle functional differences that impact migratory cell behavior.