Role of ACTN4 in Tumorigenesis, Metastasis, and EMT

Seminars in cell & developmental biology, 2017

Metastatic cancer cells invading through dense tumor stroma experience internal and external forces that are sensed through a variety of mechanosensory proteins that drive adaptations for specific environments. Alpha-actinin-4 (ACTN4) is a member of the α-actinin family of actin crosslinking proteins that is upregulated in several types of cancers. It shares 86% protein similarity with α-actinin-1, another non-muscle ACTN isoform, which appears to have a more modest role, if any, in cancer progression. While they share regulatory mechanisms, such as phosphorylation, calcium binding, phosphatidyl inositol binding, and calpain cleavage, α-actinin-4 exhibits a unique mechanosensory regulation that α-actinin-1 does not. This behavior is mediated, at least in part, by each protein's actin-binding affinity as well as the catch-slip-bond behavior of the actin binding domains. We will discuss currently known modes of ACTN4 regulation, their interactions, and how mechanosensation may pro...

Oncogene, 2000

a-Actinins are actin-binding proteins important in organization of the cytoskeleton and in cell adhesion. We have cloned and characterized a cDNA from human neuroblastoma cell variants which encodes the second non-muscle a-actinin isoform designated ACTN4 (actinin-4). mRNA encoded by the ACTN4 gene, mapped to chromosome 4, is abundant in non-tumorigenic, substrate-adherent human neuroblastoma cell variants but absent or only weakly expressed in malignant, poorly substrate-adherent neuroblasts. It is also present in many adherent tumor cell lines of diverse tissue origins. Cell lines typically co-express ACTN4 and ACTN1, a second non-muscle a-actinin gene. Expression is correlated with substrate adhesivity. Analysis of deduced amino acid sequences suggests that the two isoforms may dier in function and in regulation by calcium. Moreover, ACTN4 exhibits tumor suppressor activity. Stable clones containing increased levels of a-actinin, isolated from highly malignant neuroblastoma stem cells [BE(2)-C] after transfection with a full-length ACTN4 cDNA, show decreased anchorage-independent growth ability, loss of tumorigenicity in nude mice, and decreased expression of the N-myc proto-oncogene. Oncogene (2000) 19, 380 ± 386.

Journal of Biological Chemistry, 2011

Alpha actinins (ACTNs) are known for their ability to modulate cytoskeletal organization and cell motility by cross-linking actin filaments. We show here that ACTN4 harbors a functional LXXLL receptor interaction motif, interacts with nuclear receptors in vitro and in mammalian cells, and potently activates transcription mediated by nuclear receptors. Whereas overexpression of ACTN4 potentiates estrogen receptor ␣ (ER␣)-mediated transcription in transient transfection reporter assays, knockdown of ACTN4 decreases it. In contrast, histone deacetylase 7 (HDAC7) inhibits estrogen receptor ␣ (ER␣)-mediated transcription. Moreover, the ACTN4 mutant lacking the CaM (calmodulin)-like domain that is required for its interaction with HDAC7 fails to activate transcription by ER␣. Chromatin immunoprecipitation (ChIP) assays demonstrate that maximal associations of ACTN4 and HDAC7 with the pS2 promoter are mutually exclusive. Knockdown of ACTN4 significantly decreases the expression of ER␣ target genes including pS2 and PR and also affects cell proliferation of MCF-7 breast cancer cells with or without hormone, whereas knockdown of HDAC7 exhibits opposite effects. Interestingly, overexpression of wild-type ACTN4, but not the mutants defective in interacting with ER␣ or HDAC7, results in an increase in pS2 and PR mRNA accumulation in a hormone-dependent manner. In summary, we have identified ACTN4 as a novel, atypical coactivator that regulates transcription networks to control cell growth.

Cancer Research, 2010

A key cellular process associated with the invasive or metastatic program in many cancers is the transformation of epithelial cells toward a mesenchymal state, a process called epithelial to mesenchymal transition or EMT. Actin-dependent protrusion of cell pseudopodia is a critical element of mesenchymal cell migration and therefore of cancer metastasis. However, whether EMT occurs in human cancers and, in particular, whether it is a prerequisite for tumor cell invasion and metastasis, remains a subject of debate. Microarray and proteomic analysis of actin-rich pseudopodia from six metastatic human tumor cell lines identified 384 mRNAs and 64 proteins common to the pseudopodia of six metastatic human tumor cell lines of various cancer origins leading to the characterization of 19 common pseudopod-specific proteins. Four of these (AHNAK, septin-9, eIF4E, and S100A11) are shown to be essential for pseudopod protrusion and tumor cell migration and invasion. Knockdown of each of these p...

PLoS ONE, 2010

Background: a-Actinins cross-link actin filaments, with this cross-linking activity regulating the formation of focal adhesions, intracellular tension, and cell migration. Most non-muscle cells such as fibroblasts express two isoforms, a-actinin-1 (ACTN1) and a-actinin-4 (ACTN4). The high homology between these two isoforms would suggest redundancy of their function, but recent studies have suggested different regulatory roles. Interestingly, ACTN4 is phosphorylated upon growth factor stimulation, and this loosens its interaction with actin. Methodology/Principal Findings: Using molecular, biochemical and cellular techniques, we probed the cellular functions of ACTN4 in fibroblasts. Knockdown of ACTN4 expression in murine lung fibroblasts significantly impaired cell migration, spreading, adhesion, and proliferation. Surprisingly, knockdown of ACTN4 enhanced cellular compaction and contraction force, and increased cellular and nuclear cross-sectional area. These results, except the increased contractility, are consistent with a putative role of ACTN4 in cytokinesis. For the transcellular tension, knockdown of ACTN4 significantly increased the expression of myosin light chain 2, a element of the contractility machinery. Re-expression of wild type human ACTN4 in ACTN4 knockdown murine lung fibroblasts reverted cell spreading, cellular and nuclear cross-sectional area, and contractility back towards baseline, demonstrating that the defect was due to absence of ACTN4. Significance: These results suggest that ACTN4 is essential for maintaining normal spreading, motility, cellular and nuclear cross-sectional area, and contractility of murine lung fibroblasts by maintaining the balance between transcellular contractility and cell-substratum adhesion.

Cancer Research, 2006

Carcinoma cell motility and invasion are prerequisites for tumor cell metastasis, which requires regulation of the actin cytoskeleton. Cortactin is an actin-related protein 2/3 (Arp2/3) complex–activating and filamentous (F)-actin–binding protein that is implicated in tumor cell motility and metastasis, partially by its ability to become tyrosine phosphorylated. Cortactin is encoded by the CTTN gene and maps to chromosome 11q13, a region amplified in many carcinomas, including head and neck squamous cell carcinoma (HNSCC). CTTN gene amplification is associated with lymph node metastasis and poor patient outcome, and cortactin overexpression enhances motility in tumor cells lacking 11q13 amplification. However, a direct link between increased motility and invasion has not been reported in tumor cells with chromosome 11q13 amplification and cortactin overexpression. In this study, we have examined the relationship between CTTN amplification and tumor cell motility in HNSCC. In 11 of 3...

The International Journal of Biochemistry & Cell Biology, 2004

Cell motility is crucial for tissue formation and for development of organisms. Later on cell migration remains essential throughout the lifetime of the organism for wound healing and immune responses. The actin cytoskeleton is the cellular engine that drives cell motility downstream of a complex signal transduction cascade. The basic molecular machinery underlying the assembly and disassembly of actin filaments consists of a variety of actin binding proteins that regulate the dynamic behavior of the cytoskeleton in response to different signals. The multitude of proteins and regulatory mechanisms partaking in this system makes it vulnerable to mutations and alterations in expression levels that ultimately may cause diseases. The most familiar one is cancer that in later stages is characterized by active aberrant cell migration. Indeed tumor invasion and metastasis are increasingly being associated with deregulation of the actin system.

Cancer research, 1984

The colonic epithelium in vivo is a highly indented sheet one cell thick. Culture methods have been developed to allow the normal cellular migration of the cells comprising this sheet to flatten it into a patch on the surface of a Petri dish [Friedman, E. A., Higgins, P.J., Lipkin , M., Shinya , H., and Gelb , A.M., In Vitro (Rockville), 17: 632-644, 1981]. Actin cytoskeletal organization was analyzed in such epithelial "patches" derived from several human colonic adenocarcinomas and their precursors, adenomas (benign tumors). The actin cytoskeleton was visualized by fluorescence microscopy after the fixed, permeabilized cells were stained with rhodamine-conjugated phalloidin. This drug has a very high affinity for actin filaments and a much lower affinity for monomeric actin. Actin organization was scored from 0 (no cables) to 5 points (extensive intercellular cable network). The phalloidin-stained actin found in seven adenocarcinomas had a predominantly granular fluoresc...

Cytoskeleton, 2013

Tumor initiation and progression are accompanied by complex changes in the cytoarchitecture that at the cellular level involve remodeling of the cytoskeleton. We report on the impact of a mutant b-actin (G245Dactin) on cell structure and multicellular assembly properties. To appreciate the effects of the Gly245Asp substitution on the organization of the actin cytoskeleton, we examined the polymerization properties of G245D-actin in vitro by pyrene polymerization assays and total internal reflection fluorescence microscopy (TIRF). The mutant actin on its own has a significantly reduced polymerization efficiency compared to native actin but also modifies the polymerization of actin in copolymerization experiments. Comparison of the structure of Rat-2 fibroblasts and a stably transfected derivate called Rat-2-sm9 revealed the effects of G245D-actin in a cellular environment. The overall actin levels in Rat-2-sm9 show a 1.6-fold increase with similar amounts of mutant and wild-type actin. G245D-actin expression renders Rat-2-sm9 cells highly tumorigenic in nude mice. In Rat-2-sm9 monolayers, G245D-actin triggers the formation of extensive membrane ruffles, which is a characteristic feature of many transformed cells. To approximate complex cell-cell and cell-matrix interactions that occur in tumors and might modulate the effects of G245D-actin, we extended our studies to scaffold-free 3D spheroid cultures. Bright field and scanning electron microscopy (SEM) show that Rat-2-sm9 and Rat-2 cells share essential features of spheroid formation and compaction. However, the resulting spheroids exhibit distinct phenotypes that differ mainly in surface structure and size.

Experimental Cell Research, 2015

Invadopodia are actin-rich protrusions formed by mesenchymally migrating cancer cells. They are mainly composed of actin, actin-associated proteins, integrins and proteins of signaling machineries. These protrusions display focalized proteolytic activity towards the extracellular matrix. It is well known that polymerized (F-)actin is present in these structures, but the nature of the actin isoform has not been studied before. We here show that both cytoplasmic actin isoforms, βand γ-actin, are present in the invadopodia of MDA-MB-231 breast cancer cells cultured on a 2D-surface, where they colocalize with the invadopodial marker cortactin. Invadopodial structures formed by the cells in a 3D-collagen matrix also contain βand γ-actin. We demonstrate this using isoform-specific antibodies and expression of fluorescently-tagged actin isoforms. Additionally, using simultaneous expression of differentially tagged βand γ-actin in cells, we show that the actin isoforms are present together in a single invadopodium. Cells with an increased level of βor γ-actin, display a similar increase in the number and size of invadopodia in comparison to control cells. Moreover, increasing the level of either actin isoforms also increases invasion velocity.