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Aim: The aim of this study was to clarify the significance of DNA methylation alterations of cryptogenic hepatocellular carcinomas (HCCs). Methods: Using the Infinium assay, we performed genome‐wide DNA methylation analysis of 250 liver tissue samples, including noncancerous liver tissue (U‐N) and corresponding cancerous tissue (U‐T) from patients with cryptogenic HCC without a history of excessive alcohol use and hepatitis virus infection, and whose U‐N samples showed no remarkable histological features (no microscopic evidence of simple steatosis, any form of hepatitis including nonalcoholic steatohepatitis, or liver cirrhosis). Results: We identified 3272 probes that showed significant differences of DNA methylation levels between U‐N and normal liver tissue samples from patients without HCC, indicating that a distinct DNA methylation profile had already been established at the precancerous U‐N stage. U‐Ns have a DNA methylation profile differing from that of noncancerous liver tissue of patients with nonalcoholic steatohepatitis‐related, viral hepatitis‐related, and alcoholic liver disease‐related HCCs. Such DNA methylation alterations in U‐Ns were inherited by U‐Ts. The U‐Ns showed DNA methylation alteration of ADCY5, resulting in alteration of its mRNA expression, whereas noncancerous liver tissue of patients with nonalcoholic steatohepatitis‐, viral hepatitis‐, or alcoholic liver disease‐related HCCs did not. DNA methylation levels of MICAL2 and PLEKHG2 in U‐Ts were correlated with larger tumor diameter and portal vein involvement, respectively. Conclusions: U‐N‐specific DNA hypermethylation of ADCY5 may have significance, even from the precancerous stage in liver showing no remarkable histological features. DNA hypomethylation of MICAL2 and PLEKHG2 may determine the clinicopathological features of cryptogenic HCC. [ABSTRACT FROM AUTHOR]

MICAL2 is an actin-regulatory protein that functions through redox modification of actin. Nuclear localized MICAL2 triggers the disassembly of nuclear actin, which subsequently leads to nuclear retention of the actin-binding transcriptional coregulator myocardin-related transcription factor-A (MRTF-A), which leads to the activation of serum response factor (SRF)/MRTF-A-dependent gene transcription. In this study, we show that the secreted signaling protein GAS6 (growth-arrest specific 6) and its cognate receptor Axl, a transmembrane tyrosine kinase, also induce the activation of SRF/MRTF-A and their downstream target genes. We find that serum-induced SRF/MRTF-A-dependent gene expression can be blocked, in part, by the inhibition of Axl signaling. Furthermore, we find that Gas6/Axl-induced SRF/MRTF-A-dependent transcription is dependent on MICAL2. Gas6/Axl promotes cell invasion, which is blocked by MICAL2 knockdown, suggesting that MICAL2 promotes cytoskeletal effects of the Gas6/Axl pathway. We find that Gas/6/Axl signaling promotes the nuclear localization of MICAL2, which may contribute to the ability of Gas6/SRF to augment SRF/MRTF-A-dependent gene transcription. The physiological significance of the Gas6/Axl-MICAL2 signaling pathway described here is supported by the marked gene expression correlation across a broad array of different cancers between MICAL2 and Axl and Gas6, as well as the coexpression of these genes and the known SRF/MRTF-A target transcripts. Overall, these data reveal a new link between Gas6/Axl and SRF/MRTF-A-dependent gene transcription and link MICAL2 as a novel effector of the Gas6/Axl signaling pathway. [ABSTRACT FROM AUTHOR]

Cellular form and function are controlled by the assembly and stability of actin cytoskeletal structures--but disassembling/pruning these structures is equally essential for the plasticity and remodeling that underlie behavioral adaptations. Importantly, the mechanisms of actin assembly have been well-defined--including that it is driven by actin's polymerization into filaments (F-actin) and then often bundling by crosslinking proteins into stable higher-order structures. In contrast, it remains less clear how these stable bundled F-actin structures are rapidly disassembled. We now uncover mechanisms that rapidly and extensively disassemble bundled F-actin. Using biochemical, structural, and imaging assays with purified proteins, we show that F-actin bundled with one of the most prominent crosslinkers, fascin, is extensively disassembled by Mical, the F-actin disassembly enzyme. Furthermore, the product of this Mical effect, Mical-oxidized actin, is poorly bundled by fascin, thereby further amplifying Mical's disassembly effects on bundled F-actin. Moreover, another critical F-actin regulator, cofilin, also affects fascin-bundled filaments, but we find herein that it synergizes with Mical to dramatically amplify its disassembly of bundled F-actin compared to the sum of their individual effects. Genetic and high-resolution cellular assays reveal that Mical also counteracts crosslinking proteins/bundled F-actin in vivo to control cellular extension, axon guidance, and Semaphorin/Plexin cell-cell repulsion. Yet, our results also support the idea that fascin-bundling serves to dampen Mical's F-actin disassembly in vitro and in vivo--and that physiologically relevant cellular remodeling requires a fine-tuned interplay between the factors that build bundled F-actin networks and those that disassemble them. [ABSTRACT FROM AUTHOR]

Abstract Background Gastric cancer is a common and lethal human malignancy worldwide and cancer cell metastasis is the leading cause of cancer-related mortality. MICAL2, a flavoprotein monooxygenase, is an important regulator of epithelial-to-mesenchymal transition. The aim of this study was to explore the effects of MICAL2 on gastric cancer cell migration and determine the underlying molecular mechanisms. Methods Cell migration was examined by wound healing and transwell assays. Changes in E-cadherin/β-catenin signaling were determined by qPCR and analysis of cytoplasmic and nuclear protein fractions. E-cadherin/β-catenin binding was determined by co-immunoprecipitation assays. Cdc42 activity was examined by pulldown assay. Results MICAL2 was highly expressed in gastric cancer tissues. The knockdown of MICAL2 significantly attenuated migratory ability and β-catenin nuclear translocation in gastric cancer cells while LiCl treatment, an inhibitor of GSK3β, reversed these MICAL2 knockdown-induced effects. Meanwhile, E-cadherin expression was markedly enhanced in MICAL2-depleted cells. MICAL2 knockdown led to a significant attenuation of E-cadherin ubiquitination and degradation in a Cdc42-dependent manner, then enhanced E-cadherin/β-catenin binding, and reduced β-catenin nuclear translocation. Conclusions Together, our results indicated that MICAL2 promotes E-cadherin ubiquitination and degradation, leading to enhanced β-catenin signaling via the disruption of the E-cadherin/β-catenin complex and, consequently, the promotion of gastric cell migration. Video Abstract

Actin and its dynamic structural remodelings are involved in multiple cellular functions, including maintaining cell shape and integrity, cytokinesis, motility, navigation, and muscle contraction. Many actin-binding proteins regulate the cytoskeleton to facilitate these functions. Recently, actin’s post-translational modifications (PTMs) and their importance to actin functions have gained increasing recognition. The MICAL family of proteins has emerged as important actin regulatory oxidation-reduction (Redox) enzymes, influencing actin’s properties both in vitro and in vivo. MICALs specifically bind to actin filaments and selectively oxidize actin’s methionine residues 44 and 47, which perturbs filaments’ structure and leads to their disassembly. This review provides an overview of the MICALs and the impact of MICAL-mediated oxidation on actin’s properties, including its assembly and disassembly, effects on other actin-binding proteins, and on cells and tissue systems.

Background: Gastric cancer is a common and lethal human malignancy worldwide and cancer cell metastasis is the leading cause of cancer-related mortality. MICAL2, a flavoprotein monooxygenase, is an important regulator of epithelial-to-mesenchymal transition. The aim of this study was to explore the effects of MICAL2 on gastric cancer cell migration and determine the underlying molecular mechanisms. Methods: Cell migration was examined by wound healing and transwell assays. Changes in E-cadherin/β-catenin signaling were determined by qPCR and analysis of cytoplasmic and nuclear protein fractions. E-cadherin/β-catenin binding was determined by co-immunoprecipitation assays. Cdc42 activity was examined by pulldown assay. Results: MICAL2 was highly expressed in gastric cancer tissues. The knockdown of MICAL2 significantly attenuated migratory ability and β-catenin nuclear translocation in gastric cancer cells while LiCl treatment, an inhibitor of GSK3β, reversed these MICAL2 knockdown-induced effects. Meanwhile, E-cadherin expression was markedly enhanced in MICAL2-depleted cells. MICAL2 knockdown led to a significant attenuation of E-cadherin ubiquitination and degradation in a Cdc42-dependent manner, then enhanced E-cadherin/β-catenin binding, and reduced β-catenin nuclear translocation. Conclusions: Together, our results indicated that MICAL2 promotes E-cadherin ubiquitination and degradation, leading to enhanced β-catenin signaling via the disruption of the E-cadherin/β-catenin complex and, consequently, the promotion of gastric cell migration. Dc64RryusdU76Z1k9R6nBL Video Abstract [ABSTRACT FROM AUTHOR]

Recent studies showed that molecule interacting with CasL2 (MICAL2) could be a novel tumor growth factor, and it is closely associated with tumor growth and invasion. However, the role it plays in glioblastoma (GBM) and its potential mechanisms are currently unknown. Our study is designed to identify the effect of MICAL2 on GBM cells and the potential mechanisms behind it. Here, we found that MICAL2 interacts with TGF receptor-type I (TGFRI) and promotes the proliferation and migration of glioblastoma through the TGF-β/p-Smad2/EMT-like signaling pathway. MICAL2-knockdown inhibited the proliferation of glioblastoma cells, which was related to cell cycle arrest and downregulation of DNA replication. The invasion abilities of U87 and U251 cells were reduced after the knockdown of MICAL2. MICAL2 promoted the growth of GBM in nude mice. High MICAL2 predicts poor outcome of GBM patients. MICAL2 could be identified as a novel promising therapeutic target for human GBM. [ABSTRACT FROM AUTHOR]

Recent studies showed that molecule interacting with CasL2 (MICAL2) could be a novel tumor growth factor, and it is closely associated with tumor growth and invasion. However, the role it plays in glioblastoma (GBM) and its potential mechanisms are currently unknown. Our study is designed to identify the effect of MICAL2 on GBM cells and the potential mechanisms behind it. Here, we found that MICAL2 interacts with TGF receptor-type I (TGFRI) and promotes the proliferation and migration of glioblastoma through the TGF-β/p-Smad2/EMT-like signaling pathway. MICAL2-knockdown inhibited the proliferation of glioblastoma cells, which was related to cell cycle arrest and downregulation of DNA replication. The invasion abilities of U87 and U251 cells were reduced after the knockdown of MICAL2. MICAL2 promoted the growth of GBM in nude mice. High MICAL2 predicts poor outcome of GBM patients. MICAL2 could be identified as a novel promising therapeutic target for human GBM.

Aims and Hypothesis: Cell migration is driven by the reorganization of the actin cytoskeleton. Although MICAL2 is known to mediate the oxidation of actin filaments to regulate F-actin dynamics, relatively few studies have investigated the potential role of MICAL2 during cancer cell migration.Methods: The migratory ability of gastric cancer cells was measured by wound healing and transwell assays. The relationship between MICAL2 expression and MRTF-A nuclear localization was analyzed using gene overexpression and knockdown strategies. The production of reactive oxygen species (ROS) was evaluated by DCFH-DA staining. mRNA and protein levels of MMP9 were measured using qPCR and immunoblotting analysis. The activities of CDC42 and RhoA were assessed using pulldown assays.Results: Depletion of MICAL2 markedly reduced gastric cancer cell migration. Mechanistically, silencing of MICAL2 inhibited the nuclear translocation of MRTF-A in response to EGF and serum stimulation, whereas the contents of MRTF-A remained unchanged. Further analysis showed that silencing of MICAL2 decreased the activation of CDC42 as well as mRNA and protein levels of MMP9. Ectopic expression of MICAL2 augmented MRTF-A levels in the nucleus, and promoted the activation of CDC42, MMP9 expression, and gastric cancer cell migration. Moreover, silencing of MRTF-A inhibited the CDC42 activation induced by overexpression of MICAL2. In addition, MICAL2-induced ROS generation contributed to the effect exerted by MICAL2 on MRTF-A nuclear translocation.Conclusion: Together, these results provide evidence that MICAL2 facilitates gastric cancer cell migration via positive regulation of nuclear translocation of MRTF-A and subsequent CDC42 activation and MMP9 expression.

Molecule interacting with CasL 2 (MICAL2), a cytoskeleton dynamics regulator, are strongly expressed in several human cancer types, especially at the invasive front, in metastasizing cancer cells and in the neo-angiogenic vasculature. Although a plethora of data exist and stress a growing relevance of MICAL2 to human cancer, it is worth noting that only one small-molecule inhibitor, named CCG-1423 (1), is known to date. Herein, with the aim to develop novel MICAL2 inhibitors, starting from CCG-1423 (1), a small library of new compounds was synthetized and biologically evaluated on human dermal microvascular endothelial cells (HMEC-1) and on renal cell adenocarcinoma (786-O) cells. Among the novel compounds, 10 and 7 gave interesting results in terms of reduction in cell proliferation and/or motility, whereas no effects were observed in MICAL2-knocked down cells. Aside from the interesting biological activities, this work provides the first structure–activity relationships (SARs) of CCG-1423 (1), thus providing precious information for the discovery of new MICAL2 inhibitors.

The capacity of inducing angiogenesis is a recognized hallmark of cancer cells. The cancer microenvironment, characterized by hypoxia and inflammatory signals, promotes proliferation, migration and activation of quiescent endothelial cells (EC) from surrounding vascular network. Current anti-angiogenic drugs present side effects, temporary efficacy, and issues of primary resistance, thereby calling for the identification of new therapeutic targets. MICALs are a unique family of redox enzymes that destabilize F-actin in cytoskeletal dynamics. MICAL2 mediates Semaphorin3A-NRP2 response to VEGFR1 in rat ECs. MICAL2 also enters the p130Cas interactome in response to VEGF in HUVEC. Previously, we showed that MICAL2 is overexpressed in metastatic cancer. A small-molecule inhibitor of MICAL2 exists (CCG-1423). Here we report that 1) MICAL2 is expressed in neo-angiogenic ECs in human solid tumors (kidney and breast carcinoma, glioblastoma and cardiac myxoma, n = 67, were analyzed with immunohistochemistry) and in animal models of ischemia/inflammation neo-angiogenesis, but not in normal capillary bed; 2) MICAL2 protein pharmacological inhibition (CCG-1423) or gene KD reduce EC viability and functional performance; 3) MICAL2 KD disables ECs response to VEGF in vitro. Whole-genome gene expression profiling reveals MICAL2 involvement in angiogenesis and vascular development pathways. Based on these results, we propose that MICAL2 expression in ECs participates to inflammation-induced neo-angiogenesis and that MICAL2 inhibition should be tested in cancer- and noncancer-associated neo-angiogenesis, where chronic inflammation represents a relevant pathophysiological mechanism. Unlabelled Image • MICAL2 is expressed in human cancer- and ischemia/inflammation-associated neo-angiogenic endothelial cells. • MICAL2 is not expressed in normal capillaries. • TNF-alpha strongly increases MICAL2 expression. • MICAL2 inhibition reduces endothelial cells viability and functions, making them refractory to VEGF. • Transcriptome analysis confirmed MICAL2 contribution in angiogenesis pathways. [ABSTRACT FROM AUTHOR]

Pulmonary arterial hypertension (PAH) is a devastating and fatal vascular disease for which currently there is no satisfying therapy available. Excessive cell proliferation of pulmonary arterial smooth muscle cells (PASMCs) contributes significantly to PAH pathogenesis. In this study, we found that miR-205-5p was lowly expressed in hypoxia-induced PAH mouse model and hypoxia-treated PASMCs. Restoration of miR-205-5p suppressed PASMCs proliferation. In contrast, molecule interacting with CasL 2 (MICAL2) was highly expressed in hypoxia-induced PAH mouse model and hypoxia-treated PASMCs. Overexpression of MICAL2 promoted cell proliferation. Furthermore, miR-205-5p inhibited MICAL2 expression levels by targeting the MICAL2 3′ untranslated region. In addition, MICAL2 activated ERK1/2 signaling in PASMCs and ERK1/2 inhibitor blocked MICAL2-mediated-promotion effect on PASMCs proliferation. These results demonstrated that miR-205-5p suppressed PASMCs proliferation by targeting MICAL2, which activated ERK1/2 signaling. Therefore, miR-205-5p/MICAL2/Erk1/2 may serve as an ideal therapeutic target to PAH treatment. Unlabelled Image • miR-205-5p suppresses PASMCs proliferation. • MICAL2 promotes PASMCs proliferation. • miR-205-5p targets the 3′-UTR of MICAL2. • miR-205-5p/MICAL2/Erk 1/2 axis regulates PASMCs proliferation. [ABSTRACT FROM AUTHOR]

Abstract: Aim: MICAL2, a cytoskeleton dynamics regulator, is identified associated with survival and metastasis of several types of cancers recently. This study was designed to investigate the role of MICAL2 in breast cancer cell migration as well as its underlying mechanisms. Methods: The relationship between MICAL2 and EGF/EGFR signalling was analysed by gene overexpression and knock‐down techniques. Cell migration was measured by wound‐healing assays. Activation of EGF/EGFR signalling pathways were evaluated by immunofluorescence, qPCR, Western blotting and zymography techniques. Rac1 activity was assessed by pull‐down assay. Correlation of MICAL2 and EGFR in breast cancer specimens was examined by immunohistochemical analysis. Results: Ectopic expression of MICAL2 in MCF‐7 cells augmented EGFR protein level, accompanied by the promotion of cell migration. Silencing MICAL2 in MDA‐MB‐231 cells destabilized EGFR and inhibited cell migration. In mechanism, the maintaining effect of MICAL2 on EGFR protein content was due to a delay in EGFR degradation. Expression of MICAL2 was also shown positively correlated with the activation of P38/HSP27 and P38/MMP9 signallings, which are the main downstream signalling cascades of EGF/EGFR involved in cell migration. Further analysis indicated that Rac1 activation contributed to the maintaining effect of MICAL2 on EGFR stability. In addition, analysis of breast cancer specimens revealed a positive correlation between MICAL2 and EGFR levels and an association between MICAL2 expression and worse prognosis. Conclusion: MICAL2 is a major regulator of breast cancer cell migration, maintaining EGFR stability and subsequent EGFR/P38 signalling activation through inhibiting EGFR degradation in a Rac1‐dependent manner. [ABSTRACT FROM AUTHOR]

Actin and its dynamic structural remodelings are involved in multiple cellular functions, including maintaining cell shape and integrity, cytokinesis, motility, navigation, and muscle contraction. Many actin-binding proteins regulate the cytoskeleton to facilitate these functions. Recently, actin's post-translational modifications (PTMs) and their importance to actin functions have gained increasing recognition. The MICAL family of proteins has emerged as important actin regulatory oxidation-reduction (Redox) enzymes, influencing actin's properties both in vitro and in vivo . MICALs specifically bind to actin filaments and selectively oxidize actin's methionine residues 44 and 47, which perturbs filaments' structure and leads to their disassembly. This review provides an overview of the MICALs and the impact of MICAL-mediated oxidation on actin's properties, including its assembly and disassembly, effects on other actin-binding proteins, and on cells and tissue systems., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Rajan, Terman and Reisler.)

Summary The Drosophila melanogaster MICAL protein is essential for the neuronal growth cone machinery that functions through plexin- and semaphorin-mediated axonal signaling. Drosophila MICAL is also involved in regulating myofilament organization and synaptic structures, and serves as an actin disassembly factor downstream of plexin-mediated axonal repulsion. In mammalian cells there are three known isoforms, MICAL1, MICAL2 and MICAL3, as well as the MICAL-like proteins MICAL-L1 and MICAL-L2, but little is known of their function, and information comes almost exclusively from neural cells. In this study we show that in non-neural cells human MICALs are required for normal actin organization, and all three MICALs regulate actin stress fibers. Moreover, we provide evidence that the generation of reactive oxygen species by MICAL proteins is crucial for their actin-regulatory function. However, although MICAL1 is auto-inhibited by its C-terminal coiled-coil region, MICAL2 remains constitutively active and affects stress fibers. These data suggest differential but complementary roles for MICAL1 and MICAL2 in actin microfilament regulation. [ABSTRACT FROM AUTHOR]

Molecule interacting with CasL 2 (MICAL2), a cytoskeleton dynamics regulator, are strongly expressed in several human cancer types, especially at the invasive front, in metastasizing cancer cells and in the neo-angiogenic vasculature. Although a plethora of data exist and stress a growing relevance of MICAL2 to human cancer, it is worth noting that only one small-molecule inhibitor, named CCG-1423 (1), is known to date. Herein, with the aim to develop novel MICAL2 inhibitors, starting from CCG-1423 (1), a small library of new compounds was synthetized and biologically evaluated on human dermal microvascular endothelial cells (HMEC-1) and on renal cell adenocarcinoma (786-O) cells. Among the novel compounds, 10 and 7 gave interesting results in terms of reduction in cell proliferation and/or motility, whereas no effects were observed in MICAL2-knocked down cells. Aside from the interesting biological activities, this work provides the first structure–activity relationships (SARs) of CCG-1423 (1), thus providing precious information for the discovery of new MICAL2 inhibitors. [ABSTRACT FROM AUTHOR]

To determine the role of MICAL2 in myofibroblasts differentiation and epidural fibrosis. Epidural fibrosis (EF) may develop following laminectomy and aberrant myofibroblasts differentiation and excessive extracellular matrix (ECM) accumulation play key roles in the formation of EF. Dense epidural fibrosis results to the poor surgical outcomes and failed back surgery syndrome (FBSS), and there is no effective treatment available. Molecule interacting with Casl2 (MICAL2) has been demonstrated to participate in multiple cellular processes by regulating actin cytoskeleton dynamics. However, its role in epidural fibrosis remains totally unverified. The potential functions and mechanisms of MICAL2 were explored using western blotting, immunofluorescence and lentivirus infection. In our study, we determined that the MICAL2 expression was elevated in epidural fibrotic tissues and TGF-β1-stimulated fibroblasts. Moreover, knockdown of MICAL2 using MICAL2-specific short hairpin RNA attenuated TGF-β1-induced myofibroblasts differentiation and epidural fibrosis both in vitro and vivo, as indicated by decreased scar formation, reduced collagen production and down-regulated expression of α-SMA, collagen-1 and fibronectin. We also demonstrated that MICAL2 knockdown affected the migratory capability of fibroblasts in vitro. By further mechanistic research, we revealed that the MRTF-A nuclear translocation was inhibited in response to the knockdown of MICAL2 in fibroblasts and MICAL2 served as a pro-fibrotic factor in an SRF/MRTF-A-dependent manner. In conclusion, our results indicated that MICAL2 mediated myofibroblasts differentiation and promoted epidural fibrogenesis via SRF/MRTF-A signaling pathway, suggesting manipulation of MICAL2 activity as a novel alternative strategy for the prevention of epidural fibrosis. [ABSTRACT FROM AUTHOR]

To change their behaviors, cells require actin proteins to assemble together into long polymers/filaments—and so a critical goal is to understand the factors that control this actin filament (F-actin) assembly and stability. We have identified a family of unusual actin regulators, the MICALs, which are flavoprotein monooxygenase/hydroxylase enzymes that associate with flavin adenine dinucleotide (FAD) and use the co-enzyme nicotinamide adenine dinucleotide phosphate (NADPH) in Redox reactions. F-actin is a specific substrate for these MICAL Redox enzymes, which oxidize specific amino acids within actin to destabilize actin filaments. Furthermore, this MICAL-catalyzed reaction is reversed by another family of Redox enzymes (SelR/MsrB enzymes)—thereby revealing a reversible Redox signaling process and biochemical mechanism regulating actin dynamics. Interestingly, in addition to the MICALs' Redox enzymatic portion through which MICALs covalently modify and affect actin, MICALs have multiple other domains. Less is known about the roles of these other MICAL domains. Here we provide approaches for obtaining high levels of recombinant protein for the Redox only portion of Mical and demonstrate its catalytic and F-actin disassembly activity. These results provide a ground state for future work aimed at defining the role of the other domains of Mical — including characterizing their effects on Mical's Redox enzymatic and F-actin disassembly activity. [ABSTRACT FROM AUTHOR]

Aims and Hypothesis: Cell migration is driven by the reorganization of the actin cytoskeleton. Although MICAL2 is known to mediate the oxidation of actin filaments to regulate F-actin dynamics, relatively few studies have investigated the potential role of MICAL2 during cancer cell migration. Methods: The migratory ability of gastric cancer cells was measured by wound healing and transwell assays. The relationship between MICAL2 expression and MRTF-A nuclear localization was analyzed using gene overexpression and knockdown strategies. The production of reactive oxygen species (ROS) was evaluated by DCFH-DA staining. mRNA and protein levels of MMP9 were measured using qPCR and immunoblotting analysis. The activities of CDC42 and RhoA were assessed using pulldown assays. Results: Depletion of MICAL2 markedly reduced gastric cancer cell migration. Mechanistically, silencing of MICAL2 inhibited the nuclear translocation of MRTF-A in response to EGF and serum stimulation, whereas the contents of MRTF-A remained unchanged. Further analysis showed that silencing of MICAL2 decreased the activation of CDC42 as well as mRNA and protein levels of MMP9. Ectopic expression of MICAL2 augmented MRTF-A levels in the nucleus, and promoted the activation of CDC42, MMP9 expression, and gastric cancer cell migration. Moreover, silencing of MRTF-A inhibited the CDC42 activation induced by overexpression of MICAL2. In addition, MICAL2-induced ROS generation contributed to the effect exerted by MICAL2 on MRTF-A nuclear translocation. Conclusion: Together, these results provide evidence that MICAL2 facilitates gastric cancer cell migration via positive regulation of nuclear translocation of MRTF-A and subsequent CDC42 activation and MMP9 expression., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Wang, Min, Qi, Zhao, Yu, Zhang and Du.)

Molecule interacting with CasL2 (MICAL2), a microtubule-associated monooxygenase, is highly expressed in various cancers and is involved in cancer pathogenesis, but the mechanisms underlying its regulation in carcinogenesis are unclear. In this study, we aim to clarify the mechanism by which MICAL2 participates in colorectal cancer (CRC) and identify novel markers for predicting prognosis of CRC patients. Methods: The value of MICAL2 in CRC prognosis was determined by immunohistochemical analysis of a CRC biopsy array. A short hairpin RNA target MICAL2 (shMICAL2) was designed to knock down MICAL2 expression and observe MICAL2's function on CRC cell growth. mRNA expression array was used to screen target molecules of MICAL2. HCT116 p53 +/+ and HCT116 p53 -/- cells were used to confirm whether MICAL2 exerts its oncogenic effect through p53. The in vivo effect of MICAL2 on CRC growth was assessed by subcutaneously injecting MICAL2 -knockout CRC cells into the dorsal flank of each mouse. Immunofluorescence was used to observe the effect of MICAL2 on p53 cellular location. Reverse-phase nano ESI-LCMS analysis was used to investigate if MICAL2 mediates p53 oxidation. Results: MICAL2 was found to be highly expressed in CRC tissues, and its expression was associated with CRC carcinogenesis and poor patient outcome. MICAL2- knockdown decreased growth and colony formation of CRC cells, which was linked with cell cycle arrest and apoptosis. MICAL2 physically interacted with p53 and retained p53 in the cytoplasm. MICAL2 shortened the half-life of p53, and ectopic MICAL2 expression decreased p53 protein stability through ubiquitin degradation. MICAL2 was also found to oxidize p53 at methionine 40 and 160, which mediated p53 ubiquitin degradation. MICAL2-promoted CRC growth in vivo was confirmed in nude mice. Conclusion: MICAL2 binds to p53, retains p53 in the cytoplasm and oxidizes it at Met 40 and 160, promotes p53 ubiquitination, and decreases p53 function. MICAL2-reduced p53 promotes CRC development., Competing Interests: Competing Interests: The authors have declared that no competing interest exists.

Molecule interacting with CasL 2 (MICAL2), a microtubule associated monooxygenase, is involved in cell growth, axon guidance, vesicle trafficking and apoptosis. Recent studies have demonstrated that MICAL2 is highly expressed in tumor and accelerates tumor progression and it is deemed to be a novel tumor-promoting factor. MICAL2 overexpression increases cell proliferation to accelerate tumor growth, and MICAL2 also promotes epithelial-mesenchymal transition (EMT)-related proteins to increase cancer cell metastasis. On mechanism, MICAL2 induces EMT by regulating SRF (serum response factor)/MRTF-A (myocardin related transcription factor A) signaling, Semaphorin/Plexin pathway and inducing ROS (Reactive oxygen species) production. In the present review, we introduced MICAL family, expatiated the structure and functions of MICALs, and summarized the mechanisms of MICAL2 involving tumor progression. The challenges and perspectives for MICAL2 in tumor are also discussed., Competing Interests: Competing Interests: The authors have declared that no competing interest exists.

The MICAL (Molecules Interacting with CasL) proteins catalyze actin oxidation-reduction reactions destabilizing F-actin in cytoskeletal dynamics. Here we show for the first time that MICAL2 mRNA is significantly over-expressed in aggressive, poorly differentiated/undifferentiated, primary human epithelial cancers (gastric and renal). Immunohistochemistry showed MICAL2-positive cells on the cancer invasive front and in metastasizing cancer cells inside emboli, but not at sites of metastasis, suggesting MICAL2 expression was 'on' in a subpopulation of primary cancer cells seemingly detaching from the tissue of origin, enter emboli and travel to distant sites, and was turned 'off' upon homing at metastatic sites. In vitro, MICAL2 knock-down resulted in mesenchymal to epithelial transition, reduction of viability, and loss of motility and invasion properties of human cancer cells. Moreover, expression of MICAL2 cDNA in MICAL2-depleted cells induced epithelial to mesenchymal transition. Altogether our data indicate that MICAL2 over-expression is associated with cancer progression and metastatic disease. MICAL2 might be an important regulator of epithelial to mesenchymal transition and therefore a promising target for anti-metastatic therapy.