Genetic disruption of ATAT1 causes RhoA downregulation through abnormal truncation of C/EBPß.

Microtubule acetylation has been shown to regulate actin filament dynamics by modulating signaling pathways that control actin organization, although the precise mechanisms remain unknown. In this study, we found that the downregulation of microtubule acetylation via the disruption ATAT1 (which encodes α-tubulin N-acetyltransferase 1) inhibited the expression of RhoA, a small GTPase involved in regulating the organization of actin filaments and the formation of stress fibers. Analysis of RHOA promoter and chromatin immunoprecipitation assays revealed that C/EBPß is a major regulator of RHOA expression. Interestingly, the majority of C/EBPß in ATAT1 knockout (KO) cells was found in the nucleus as a 27-kDa fragment (referred to as C/EBPßp27) lacking the N-terminus of C/EBPß. Overexpression of a gene encoding a C/EBPßp27-mimicking protein via an N-terminal deletion in C/EBPß led to competitive binding with wild-type C/EBPß at the C/EBPß binding site in the RHOA promoter, resulting in a significant decrease of RHOA expression. We also found that cathepsin L (CTSL), which is overexpressed in ATAT1 KO cells, is responsible for C/EBPßp27 formation in the nucleus. Treatment with a CTSL inhibitor led to the restoration of RHOA expression by downregulation of C/EBPßp27 and the invasive ability of ATAT1 KO MDA-MB-231 breast cancer cells. Collectively, our findings suggest that the downregulation of microtubule acetylation associated with ATAT1 deficiency suppresses RHOA expression by forming C/EBPßp27 in the nucleus through CTSL. We propose that CTSL and C/EBPßp27 may represent a novel therapeutic target for breast cancer treatment. [BMB Reports 2024; 57(6): 293-298].

Microtubule acetylation induced by oxidative stress regulates subcellular distribution of lysosomal vesicles for amyloid-beta secretion.

Excessive production and accumulation of amyloid-beta (Aß) in the brain are one of the hallmarks of Alzheimer's disease (AD). Although oxidative stress is known to trigger and promote the progression of AD, the molecular relationship between oxidative stress and Aß production is not yet fully understood. In this study, we demonstrate that microtubule acetylation induced by oxidative stress plays a critical role in Aß production and secretion by altering the subcellular distribution of Aß precursor protein (APP)-containing lysosomal vesicles. Under oxidative stress, both H4-APPSwe/Ind and HEK293T-APPSwe/Ind cell lines showed increased microtubule acetylation and Aß secretion. Knockdown (KD) of alpha-tubulin N-acetyltransferase 1 (ATAT1) by using a lentiviral shRNA not only inhibited the generation of intermediate APP fragments, such as ß-CTF and AICD, but also suppressed Aß secretion. Oxidative stress promoted the dispersion of LAMP1-positive vesicles to the periphery of the cell through microtubule acetylation, leading to the formation of neutralized lysosomal vesicles (NLVs), which was inhibited by ATAT1 KD. Treatment of the cells with the dynein ATPase inhibitor EHNA or downregulation of LIS1, a regulator of dynein-mediated intracellular transport, increased the peripheral localization of NLVs and promoted Aß secretion, whereas KD of ADP ribosylation factor like GTPase 8B showed the opposite result. ATAT1 KD in the hippocampal region of the 5×FAD AD mouse model also showed significant reductions in Aß plaque accumulation and memory loss. Taken together, these findings suggest that oxidative stress-induced microtubule acetylation promotes the peripheral localization of lysosomal vesicles to form NLVs, thereby enhancing Aß secretion.

Downregulation of AP1S1 causes the lysosomal degradation of EGFR in non-small cell lung cancer.

Matrix stiffness has been shown to play a critical role in cancer progression by influencing various cellular processes, including epidermal growth factor (EGF) signaling. However, the underlying molecular mechanisms are not fully understood. Here, we investigated the role of adaptor-related protein complex 1 subunit sigma 1 (AP1S1), a component of adaptor protein complex-1, in the regulation of EGF receptor (EGFR) intracellular trafficking during cancer cell progression. We found that AP1S1 expression was upregulated under stiff matrix conditions, resulting in the regulation of EGFR trafficking in non-small cell lung adenocarcinoma cells. Knockout of AP1S1 caused the lysosomal degradation of EGFR, leading to suppressed EGF-induced anaplastic lymphoma receptor tyrosine kinase phosphorylation. In addition, the downregulation of AP1S1 increased the sensitivity of H1975 cancer cells, which are resistant to tyrosine kinase inhibitors, to erlotinib. Collectively, our results suggest that AP1S1 could regulate EGFR recycling under stiff matrix conditions, and AP1S1 inhibition could be a novel strategy for treating cancer cells resistant to EGFR-targeted anticancer drugs.

Casein kinase 2 promotes the TGF-ß-induced activation of α-tubulin acetyltransferase 1 in fibroblasts cultured on a soft matrix.

Cell signals for growth factors depend on the mechanical properties of the extracellular matrix (ECM) surrounding the cells. Microtubule acetylation is involved in the transforming growth factor (TGF)-ß-induced myofibroblast differentiation in the soft ECM. However, the mechanism of activation of α-tubulin acetyltransferase 1 (α-TAT1), a major α-tubulin acetyltransferase, in the soft ECM is not well defined. Here, we found that casein kinase 2 (CK2) is required for the TGF-ß-induced activation of α-TAT1 that promotes microtubule acetylation in the soft matrix. Genetic mutation and pharmacological inhibition of CK2 catalytic activity specifically reduced microtubule acetylation in the cells cultured on a soft matrix rather than those cultured on a stiff matrix. Immunoprecipitation analysis showed that CK2α, a catalytic subunit of CK2, directly bound to the C-terminal domain of α-TAT1, and this interaction was more prominent in the cells cultured on the soft matrix. Moreover, the substitution of alanine with serine, the 236th amino acid located at the C-terminus, which contains the CK2-binding site of α-TAT1, significantly abrogated the TGF-ß-induced microtubule acetylation in the soft matrix, indicating that the successful binding of CK2 and the C-terminus of α-TAT1 led to the phosphorylation of serine at the 236th position of amino acids in α-TAT1 and regulation of its catalytic activity. Taken together, our findings provide novel insights into the molecular mechanisms underlying the TGF-ß-induced activation of α-TAT1 in a soft matrix. [BMB Reports 2022; 55(4): 192-197].

A missense variant in SHARPIN mediates Alzheimer's disease-specific brain damages.

Established genetic risk factors for Alzheimer's disease (AD) account for only a portion of AD heritability. The aim of this study was to identify novel associations between genetic variants and AD-specific brain atrophy. We conducted genome-wide association studies for brain magnetic resonance imaging measures of hippocampal volume and entorhinal cortical thickness in 2643 Koreans meeting the clinical criteria for AD (n = 209), mild cognitive impairment (n = 1449) or normal cognition (n = 985). A missense variant, rs77359862 (R274W), in the SHANK-associated RH Domain Interactor (SHARPIN) gene was associated with entorhinal cortical thickness (p = 5.0 × 10-9) and hippocampal volume (p = 5.1 × 10-12). It revealed an increased risk of developing AD in the mediation analyses. This variant was also associated with amyloid-ß accumulation (p = 0.03) and measures of memory (p = 1.0 × 10-4) and executive function (p = 0.04). We also found significant association of other SHARPIN variants with hippocampal volume in the Alzheimer's Disease Neuroimaging Initiative (rs3417062, p = 4.1 × 10-6) and AddNeuroMed (rs138412600, p = 5.9 × 10-5) cohorts. Further, molecular dynamics simulations and co-immunoprecipitation indicated that the variant significantly reduced the binding of linear ubiquitination assembly complex proteins, SHPARIN and HOIL-1 Interacting Protein (HOIP), altering the downstream NF-κB signaling pathway. These findings suggest that SHARPIN plays an important role in the pathogenesis of AD.

Beta-Pix-dynamin 2 complex promotes colorectal cancer progression by facilitating membrane dynamics.

PURPOSE: Spatiotemporal regulation of cell membrane dynamics is a major process that promotes cancer cell invasion by acting as a driving force for cell migration. Beta-Pix (ßPix), a guanine nucleotide exchange factor for Rac1, has been reported to be involved in actin-mediated cellular processes, such as cell migration, by interacting with various proteins. As yet, however, the molecular mechanisms underlying ßPix-mediated cancer cell invasion remain unclear. METHODS: The clinical significance of ßPix was analyzed in patients with colorectal cancer (CRC) using public clinical databases. Pull-down and immunoprecipitation assays were employed to identify novel binding partners for ßPix. Additionally, various cell biological assays including immunocytochemistry and time-lapse video microscopy were performed to assess the effects of ßPix on CRC progression. A ßPix-SH3 antibody delivery system was used to determine the effects of the ßPix-Dyn2 complex in CRC cells. RESULTS: We found that the Src homology 3 (SH3) domain of ßPix interacts with the proline-rich domain of Dynamin 2 (Dyn2), a large GTPase. The ßPix-Dyn2 interaction promoted lamellipodia formation, along with plasma membrane localization of membrane-type 1 matrix metalloproteinase (MT1-MMP). Furthermore, we found that Src kinase-mediated phosphorylation of the tyrosine residue at position 442 of ßPix enhanced ßPix-Dyn2 complex formation. Disruption of the ßPix-Dyn2 complex by ßPix-SH3 antibodies targeting intracellular ßPix inhibited CRC cell invasion. CONCLUSIONS: Our data indicate that spatiotemporal regulation of the Src-ßPix-Dyn2 axis is crucial for CRC cell invasion by promoting membrane dynamics and MT1-MMP recruitment into the leading edge. The development of inhibitors that disrupt the ßPix-Dyn2 complex may be a useful therapeutic strategy for CRC.

Microtubule Acetylation Controls MDA-MB-231 Breast Cancer Cell Invasion through the Modulation of Endoplasmic Reticulum Stress.

During aggressive cancer progression, cancer cells adapt to unique microenvironments by withstanding various cellular stresses, including endoplasmic reticulum (ER) stress. However, the mechanism whereby cancer cells overcome the ER stress to survive remains to be elucidated. Herein, we demonstrated that microtubule acetylation in cancer cells grown on a stiff matrix promotes cancer progression by preventing excessive ER stress. Downregulation of microtubule acetylation using shRNA or CRSIPR/Cas9 techniques targeting ATAT1, which encodes α-tubulin N-acetyltransferase (αTAT1), resulted in the upregulation of ER stress markers, changes in ER morphology, and enhanced tunicamycin-induced UPR signaling in cancer cells. A set of genes involved in cancer progression, especially focal adhesion genes, were downregulated in both ATAT1-knockout and tunicamycin-treated cells, whereas ATAT1 overexpression restored the gene expression inhibited by tunicamycin. Finally, the expression of ATAT1 and ER stress marker genes were negatively correlated in various breast cancer types. Taken together, our results suggest that disruption of microtubule acetylation is a potent therapeutic tool for preventing breast cancer progression through the upregulation of ER stress. Moreover, ATAT1 and ER stress marker genes may be useful diagnostic markers in various breast cancer types.

Dynein-mediated nuclear translocation of yes-associated protein through microtubule acetylation controls fibroblast activation.

Myofibroblasts are the major cell type that is responsible for increase in the mechanical stiffness in fibrotic tissues. It has well documented that the TGF-ß/Smad axis is required for myofibroblast differentiation under the rigid substrate condition. However, the mechanism driving myofibroblast differentiation in soft substrates remains unknown. In this research, we demonstrated that interaction of yes-associated protein (YAP) and acetylated microtubule via dynein, a microtubule motor protein drives nuclear localization of YAP in the soft matrix, which in turn increased TGF-ß1-induced transcriptional activity of Smad for myofibroblast differentiation. Pharmacological and genetical disruption of dynein impaired the nuclear translocation of YAP and decreased the TGF-ß1-induced Smad activity even though phosphorylation and nuclear localization of Smad occurred normally in α-tubulin acetyltransferase 1 (α-TAT1) knockout cell. Moreover, microtubule acetylation prominently appeared in the fibroblast-like cells nearby the blood vessel in the fibrotic liver induced by CCl4 administration, which was conversely decreased by TGF-ß receptor inhibitor. As a result, quantitative inhibition of microtubule acetylation may be suggested as a new target for overcoming fibrotic diseases.

Shwachman-Bodian-Diamond syndrome protein desensitizes breast cancer cells to apoptosis in stiff matrices by repressing the caspase 8-mediated pathway.

Certain cancer types, including breast cancer, are accompanied with stiffening of the surrounding extracellular matrix (ECM). Previous studies suggest that this stiffened matrix influences cancer cell progression, such as proliferation and invasion, both biochemically and mechanically. However, the contribution of ECM stiffness to cellular response to diverse stresses, which most cancer cells are exposed to, has not been elucidated. In this study, we demonstrate that expression of the Shwachman-Bodian-Diamond syndrome protein (SDBS) in a stiff matrix protects cells from apoptosis induced by environmental stress, including anticancer drugs. Cells cultured on stiff matrices were less apoptotic process induced by serum depletion than those cultured on the soft matrix. Interestingly, knockdown (KD) of SDBS among the apoptosis-related genes significantly increased apoptosis induced by serum depletion in cells cultured in a stiff matrix. Apoptosis of SDBS KD cells in a stiff matrix was significantly inhibited by the caspase 8 inhibitor, indicating that activation of the caspase 8 pathway by SDBS KD is critical for cancer cell apoptosis in stiff matrices. Additionally, we also found that downregulation of SDBS also effectively increased cell death induced by anticancer drugs, including paclitaxel, cisplatin, and eribulin. Taken together, our findings suggest that inhibition of SDBS enhances effective chemotherapy of malignant breast cancer cells in stiff ECM environments.

Downregulation of SPIN90 promotes fibroblast activation via periostin-FAK-ROCK signaling module.

Alterations in mechanical properties in the extracellular matrix are modulated by myofibroblasts and are required for progressive fibrotic diseases. Recently, we reported that fibroblasts depleted of SPIN90 showed enhanced differentiation into myofibroblasts via increased acetylation of microtubules in the soft matrix; the mechanisms of the underlying signaling network, however, remain unclear. In this study, we determine the effect of depletion of SPIN90 on FAK/ROCK signaling modules. Transcriptome analysis of Spin90 KO mouse embryonic fibroblasts (MEF) and fibroblasts activated by TGF-ß revealed that Postn is the most significantly upregulated gene. Knockdown of Postn by small interfering RNA suppressed cell adhesion and myofibroblastic differentiation and downregulated FAK activity in Spin90 KO MEF. Our results indicate that SPIN90 depletion activates FAK/ROCK signaling, induced by Postn expression, which is critical for myofibroblastic differentiation on soft matrices mimicking the mechanical environment of a normal tissue.

Spindle pole body component 25 homolog expressed by ECM stiffening is required for lung cancer cell proliferation.

Accumulating evidence has shown that matrix stiffening in cancer tissue by the deposition of extracellular matrix (ECM) is closely related with severe tumor progression. However, much less is known about the genes affected by matrix stiffness and its signaling for cancer progression. In the current research, we investigated the differential gene expression of a non-small lung adenocarcinoma cell line, H1299, cultured under the conditions of soft (∼0.5 kPa) and stiff (∼40 kPa) matrices, mimicking the mechanical environments of normal and cancerous tissues, respectively. For integrated transcriptome analysis, the genes identified by ECM stiffening were compared with 8248 genes retrieved from The Cancer Genome Atlas Lung Adenocarcinoma (TCGA). In stiff matrix, 29 genes were significantly upregulated, while 75 genes were downregulated. The screening of hazard ratios for these genes using the Kaplan-Meier Plotter identified 8 genes most closely associated with cancer progression under the condition of matrix stiffening. Among these genes, spindle pole body component 25 homolog (SPC25) was one of the most up-regulated genes in stiff matrix and tumor tissue. Knockdown of SPC25 in H1299 cells using shRNA significantly inhibited cell proliferation with downregulation of the expression of checkpoint protein, Cyclin B1, under the condition of stiff matrix whereas the proliferation rate in soft matrix was not affected by SPC25 silencing. Thus, our findings provide novel key molecules for studying the relationship of extracellular matrix stiffening and cancer progression.

Genetic disruption of tubulin acetyltransferase, αTAT1, inhibits proliferation and invasion of colon cancer cells through decreases in Wnt1/ß-catenin signaling.

Microtubules are required for diverse cellular processes, and abnormal regulation of microtubule dynamics is closely associated with severe diseases including malignant tumors. In this study, we report that α-tubulin N-acetyltransferase (αTAT1), a regulator of α-tubulin acetylation, is required for colon cancer proliferation and invasion via regulation of Wnt1 and its downstream genes expression. Public transcriptome analysis showed that expression of ATAT1 is specifically upregulated in colon cancer tissue. A knockout (KO) of ATAT1 in the HCT116 colon cancer cell line, using the CRISPR/Cas9 system showed profound inhibition of proliferative and invasive activities of these cancer cells. Overexpression of αTAT1 or the acetyl-mimic K40Q α-tubulin mutant in αTAT1 KO cells restored the invasiveness, indicating that microtubule acetylation induced by αTAT1 is critical for HCT116 cell invasion. Analysis of colon cancer-related gene expression in αTAT1 KO cells revealed that the loss of αTAT1 decreased the expression of WNT1. Mechanistically, abrogation of tubulin acetylation by αTAT1 knockout inhibited localization of ß-catenin to the plasma membrane and nucleus, thereby resulting in the downregulation of Wnt1 and of its downstream genes including CCND1, MMP-2, and MMP-9. These results suggest that αTAT1-mediated Wnt1 expression via microtubule acetylation is important for colon cancer progression.