Alpha-actnin-4 (ACTN4) selectively affects the DNA double-strand breaks repair in non-small lung carcinoma cells.

BACKGROUND: ACTN4 is an actin-binding protein involved in many cellular processes, including cancer development. High ACTN4 expression is often associated with a poor prognosis. However, it has been identified as a positive marker for platinum-based adjuvant chemotherapy for non-small cell lung cancer (NSCLC). The goal of our study was to investigate the involvement of ACTN4 in the NSCLC cells' response to the genotoxic drugs. RESULTS: We generated H1299 cells with the ACTN4 gene knock-out (ACTN4 KO), using the CRISPR/Cas9 system. The resistance of the cells to the cisplatin and etoposide was analyzed with the MTT assay. We were also able to estimate the efficiency of DNA repair through the DNA comet assay and gamma-H2AX staining. Possible ACTN4 effects on the non-homologous end joining (NHEJ) and homologous recombination (HR) were investigated using pathway-specific reporter plasmids and through the immunostaining of the key proteins. We found that the H1299 cells with the ACTN4 gene knock-out did not show cisplatin-resistance, but did display a higher resistance to the topoisomerase II inhibitors etoposide and doxorubicin, suggesting that ACTN4 might be somehow involved in the repair of DNA strand breaks. Indeed, the H1299 ACTN4 KO cells repaired etoposide- and doxorubicin-induced DNA breaks more effectively than the control cells. Moreover, the ACTN4 gene knock-out enhanced NHEJ and suppressed HR efficiency. Supporting the data, the depletion of ACTN4 resulted in the faster assembly of the 53BP1 foci with a lower number of the phospho-BRCA1 foci after the etoposide treatment. CONCLUSIONS: Thus, we are the first to demonstrate that ACTN4 may influence the resistance of cancer cells to the topoisomerase II inhibitors, and affect the efficiency of the DNA double strand breaks repair. We hypothesize that ACTN4 interferes with the assembly of the NHEJ and HR complexes, and hence regulates balance between these DNA repair pathways.

Role of ACTN4 in Tumorigenesis, Metastasis, and EMT.

The actin-binding protein ACTN4 belongs to a family of actin-binding proteins and is a non-muscle alpha-actinin that has long been associated with cancer development. Numerous clinical studies showed that changes in ACTN4 gene expression are correlated with aggressiveness, invasion, and metastasis in certain tumors. Amplification of the 19q chromosomal region where the gene is located has also been reported. Experimental manipulations with ACTN4 expression further confirmed its involvement in cell proliferation, motility, and epithelial-mesenchymal transition (EMT). However, both clinical and experimental data suggest that the effects of ACTN4 up- or down-regulation may vary a lot between different types of tumors. Functional studies demonstrated its engagement in a number of cytoplasmic and nuclear processes, ranging from cytoskeleton reorganization to regulation of different signaling pathways. Such a variety of functions may be the reason behind cell type and cell line specific responses. Herein, we will review research progress and controversies regarding the prognostic and functional significance of ACTN4 for tumorigenesis.

Erratum: Actin-binding protein alpha-actinin 4 (ACTN4) is a transcriptional co-activator of RelA/p65 sub-unit of NF-kB.

[This corrects the article DOI: 10.18632/oncotarget.901.].

Co-expression of RelA/p65 and ACTN4 induces apoptosis in non-small lung carcinoma cells.

Alpha-actinin 4 (ACTN4) is an actin-binding protein of the spectrin superfamily. ACTN4 is found both in the cytoplasm and nucleus of eukaryotic cells. The main function of cytoplasmic ACTN4 is stabilization of actin filaments and their binding to focal contacts. Nuclear ACTN4 takes part in the regulation of gene expression following by activation of certain transcription factors, but the mechanisms of regulation are not completely understood. Our previous studies have demonstrated the interaction of ACTN4 with the RelA/p65 subunit of NF-kappaB factor and the effect on its transcriptional activity in A431 and HEK293T cells. In the present work, we investigated changes in the composition of nuclear ACTN4-interacting proteins in non-small cell lung cancer cells H1299 upon stable RELA overexpression. We showed that ACTN4 was present in the nuclei of H1299 cells, regardless of the RELA expression level. The presence of ectopic RelA/p65 in H1299 cells increased the number of proteins interacting with nuclear ACTN4. Stable expression of RELA in these cells suppressed cell proliferation, which was further affected by simultaneous ACTN4 overexpression. We detected no significant effect on cell cycle but the apoptosis rate was increased in cells with a double RELA/ACTN4 overexpression. Interestingly, when expressed individually ACTN4 promoted proliferation of lung cancer cells. Furthermore, the bioinformatics analysis of gene expression in lung cancer patients suggested that overexpression of ACTN4 correlated with poor survival prognosis. We hypothesize that the effect of RELA on proliferation and apoptosis of H1299 cells can be mediated via affecting the interactome of ACTN4.

KMT Set7/9 affects genotoxic stress response via the Mdm2 axis.

Genotoxic stress inflicted by anti-cancer drugs causes DNA breaks and genome instability. DNA double strand breaks induced by irradiation or pharmacological inhibition of Topoisomerase II activate ATM (ataxia-telangiectasia-mutated) kinase signalling pathway that in turn triggers cell cycle arrest and DNA repair. ATM-dependent gamma-phosphorylation of histone H2Ax and other histone modifications, including ubiquitnylation, promote exchange of histones and recruitment of DNA damage response (DDR) and repair proteins. Signal transduction pathways, besides DDR itself, also control expression of genes whose products cause cell cycle arrest and/or apoptosis thus ultimately affecting the sensitivity of cells to genotoxic stress. In this study, using a number of experimental approaches we provide evidence that lysine-specific methyltransferase (KMT) Set7/9 affects DDR and DNA repair, at least in part, by regulating the expression of an E3 ubiquitin ligase, Mdm2. Furthermore, we show that Set7/9 physically interacts with Mdm2. Several cancer cell lines with inverse expression of Set7/9 and Mdm2 displayed diminished survival in response to genotoxic stress. These findings are signified by our bioinformatics studies suggesting that the unleashed expression of Mdm2 in cancer patients with diminished expression of Set7/9 is associated with poor survival outcome.

Actin-binding protein alpha-actinin 4 (ACTN4) is a transcriptional co-activator of RelA/p65 sub-unit of NF-kB.

ACTN4 is an actin-binding protein that participates in cytoskeleton organisation. It resides both in the cytoplasm and nucleus and physically associates with various transcription factors. Here, we describe an effect of ACTN4 expression on transcriptional activity of the RelA/p65 subunit of NF-kB. We demonstrate that ACTN4 enhances RelA/p65-dependant expression of c-fos, MMP-3 and MMP-1 genes, but it does not affect TNC, ICAM1 and FN1 expression. Importantly, actin-binding domains of ACTN4 are not critical for the nuclear translocation and co-activation of RelA/p65- dependent transcription. Collectively, our data suggest that in the nucleus, ACTN4 functions as a selective transcriptional co-activator of RelA/p65.

Proteomic analysis of ACTN4-interacting proteins reveals it's a putative involvement in mRNA metabolism.

Alpha-actinin 4 (ACTN4) is an actin-binding protein. In the cytoplasm, ACTN4 participates in structural organisation of the cytoskeleton via cross-linking of actin filaments. Nuclear localisation of ACTN4 has also been reported, but no clear role in the nucleus has been established. In this report, we describe the identification of proteins associated with ACTN4 in the nucleus. A combination of two-dimensional gel electrophoresis (2D-GE) and MALDI-TOF mass-spectrometry revealed a large number of ACTN4-bound proteins that are involved in various aspects of mRNA processing and transport. The association of ACTN4 with different ribonucleoproteins suggests that a major function of nuclear ACTN4 may be regulation of mRNA metabolism and signaling.

RelA/NF-kappaB transcription factor associates with alpha-actinin-4.

The NF-kappaB/RelA family of transcription factors regulates inducible transcription of a large number of genes in response to diverse stimuli. Little is known, however, about the location of NF-kappaB in the cytoplasm and the transport mechanism to the nucleus. We found that NF-kappaB is associated with the actin-binding protein alpha-actinin-4. NF-kappaB and alpha-actinin-4 co-localized along actin stress fibers and in membrane lamellae in A431 cells. After a 30-min stimulation with EGF or TNF-alpha, alpha-actinin-4 and p65 were found in the nucleus. Disruption of cytoskeleton by cytochalasin D prior to treatment with TNF-alpha led to increase of p65 nuclear translocation. Antibodies to p65 subunit of NF-kappaB co-immunoprecipitated alpha-actinin-4 from A431 cell lysates and nuclear extracts, but alpha-actinin-1 and beta-actin were not found in the precipitates. Affinity chromatography experiments displayed that p65 and p50 subunits of NF-kappaB can bind to matrix-bound chicken gizzard alpha-actinin. We suggest that the alpha-actinin-4 is important for the NF-kappaB nuclear translocation and its functions inside the nucleus.

Extra-cellular matrix proteins induce re-distribution of alpha-actinin-1 and alpha-actinin-4 in A431 cells.

Alpha-actinins are actin-binding proteins of non-muscle cells, which can participate in the regulation of transcription factor activity. We describe the distribution of alpha-actinin-1 and -4 depending on different actin cytoskeleton formed as a result of cell adhesion to extracellular matrix proteins, such as fibronectin and laminin 2/4. Immunofluorescent studies show a difference in the distribution of alpha-actinin and -4. Both isoforms localise along stress-fibres, but alpha-actinin-1 localises in the perinuclear region more abundantly than alpha-actinin-4. Western blot analysis demonstrated existence of truncated forms of both isoforms. Truncated alpha-actinin-1 appears in cells spread on fibronectin or laminin. Cell spreading also correlated with more tight association of alpha-actinin-4 with chromatin. Basing on our previous finding of an interaction of alpha-actinin-4 with p65 subunit of the NF-kappaB, we checked the possible influence of immobilised ligands on its redistribution in nuclear complexes containing p65. alpha-Actinin-4 seems to be present in some but not all nuclear complexes containing p65. Immobilised ligands may affect the interaction of alpha-actinin-4/p65 complexes with chromatin. The data suggest that adhesion to extra-cellular matrix may interfere in cellular reactions mediated by alpha-actinin-1 and -4.

Gene expression profiling in frataxin deficient mice: microarray evidence for significant expression changes without detectable neurodegeneration.

Friedreich's ataxia (FRDA) is caused by reduction of frataxin levels to 5-35%. To better understand the biochemical sequelae of frataxin reduction, in absence of the confounding effects of neurodegeneration, we studied the gene expression profile of a mouse model expressing 25-36% of the normal frataxin levels, and not showing a detectable phenotype or neurodegenerative features. Despite having no overt phenotype, a clear microarray gene expression phenotype was observed. This phenotype followed the known regional susceptibility in this disease, most changes occurring in the spinal cord. Additionally, gene ontology analysis identified a clear mitochondrial component, consistent with previous findings. We were able to confirm a subset of changes in fibroblast cell lines from patients. The identification of a core set of genes changing early in the FRDA pathogenesis can be a useful tool in both clarifying the disease process and in evaluating new therapeutic strategies.