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.

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.

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.

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.

Down-regulation of diesel particulate matter-induced airway inflammation by the PDZ motif peptide of ZO-1.

Although diesel airborne particulate matter (PM2.5) has been known to play a role in many human diseases, there is no direct evidence that therapeutic drugs or proteins can diminish PM2.5-induced diseases. Nevertheless, studies examining the negative control mechanisms of PM2.5-induced diseases are critical to develop novel therapeutic medications. In this study, the consensus PDZ peptide of ZO-1 inhibited PM2.5-induced inflammatory cell infiltration, pro-inflammatory cytokine gene expression, and TEER in bronchoalveolar lavage (BAL) fluid and AM cells. Our data indicated that the PDZ domain in ZO-1 is critical for regulation of the PM2.5-induced inflammatory microenvironment. Therefore, the PDZ peptide may be a potential therapeutic candidate during PM-induced respiratory diseases.

Extra domain A-containing fibronectin expression in Spin90-deficient fibroblasts mediates cancer-stroma interaction and promotes breast cancer progression.

Cancer-associated fibroblasts (CAFs) in the tumor microenvironment play major roles in supporting cancer progression. A previous report showed that SPIN90 downregulation is correlated with CAF activation and that SPIN90-deficient CAFs promote breast cancer progression. However, the mechanisms that mediate cancer-stroma interaction and how such interactions regulate cancer progression are not well understood. Here, we show that extra domain A (EDA)-containing fibronectin (FN), FN(+)EDA, produced by mouse embryonic fibroblasts (MEFs) derived from Spin90-knockout (KO) mice increases their own myofibroblast differentiation, which facilitates breast cancer progression. Increased FN(+)EDA in Spin90-KO MEFs promoted fibril formation in the extracellular matrix (ECM) and specifically interacted with integrin α4ß1 as the mediating receptor. Moreover, FN(+)EDA expression by Spin90-KO MEFs increased proliferation, migration, and invasion of breast cancer cells. Irigenin, a specific inhibitor of the interaction between integrin α4ß1 and FN(+)EDA, significantly blocked the effects of FN(+)EDA, such as fibril formation by Spin90-KO MEFs and proliferation, migration, and invasion of breast cancer cells. In orthotopic breast cancer mouse models, irigenin injection remarkably reduced tumor growth and lung metastases. It was supported by that FN(+)EDA in assembled fibrils was accumulated in cancer stroma of human breast cancer patients in which SPIN90 expression was downregulated. Our data suggest that SPIN90 downregulation increases FN(+)EDA and promotes ECM stiffening in breast cancer stroma through an assembly of long FN(+)EDA-rich fibrils; moreover, engagement of the Integrin α4ß1 receptor facilitates breast cancer progression. Inhibitory effects of irigenin on tumor growth and metastasis suggest the potential of this agent as an anticancer therapeutic.

Transcriptome dynamics of alternative splicing events revealed early phase of apoptosis induced by methylparaben in H1299 human lung carcinoma cells.

Methylparaben is most frequently used as an antimicrobial preservative in pharmaceuticals and foods. Methylparaben has been subjected to toxicological studies owing to the increasing concern regarding its possible impact on the environment and human health. However, the cytotoxicity and underlying mechanisms of methylparaben exposure in human lung cells have not been explored. Here, we investigated the effect of methylparaben on cell cycle, apoptotic pathways, and changes in the transcriptome profiles in human lung cells. Our results demonstrate that treatment with methylparaben causes inhibition of cell growth. In addition, methylparaben induced S- and G2/M-phase arrest as a result of enhanced apoptosis. Transcriptome analysis using RNA-seq revealed that mRNA expression of ER stress- and protein misfolding-related gene sets was upregulated in methylparaben-treated group. RNA splicing- and maturation-related gene sets were significantly down-regulated by methylparaben treatment. Interestingly, RNA-seq analysis at the transcript level revealed that alternative splicing events, especially retained intron, were markedly changed by a low dose of methylparaben treatment. Altogether, these data show that methylparaben induces an early phase of apoptosis through cell cycle arrest and downregulation of mRNA maturation.

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.

Histone demethylase KDM3B regulates the transcriptional network of cell-cycle genes in hepatocarcinoma HepG2 cells.

Hepatocellular carcinoma (HCC) is the fifth most common cancer and the third most lethal cancer worldwide. Although gene mutations associated with HCC development have been intensively studied, how epigenetic factors specifically modulate the functional properties of HCC by regulating target gene expression is unclear. Here we demonstrated the overexpression of KDM3B in liver tissue of HCC patients using public RNA-seq data. Ablation of KDM3B by CRISPR/Cas9 retarded the cell cycle and proliferation of hepatocarcinoma HepG2 cells. Approximately 30% of KDM3B knockout cells exhibited mitotic spindle multipolarity as a chromosome instability (CIN) phenotype. RNA-seq analysis of KDM3B knockout revealed significantly down-regulated expression of cell cycle related genes, especially cell proliferation factor CDC123. Furthermore, the expression level of Cyclin D1 was reduced in KDM3B knockout by proteosomal degradation without any change in the expression of CCND1, which encodes Cyclin D1. The results implicate KDM3B as a crucial epigenetic factor in cell cycle regulation that manipulates chromatin dynamics and transcription in HCC, and identifies a potential gene therapy target for effective treatment of HCC.

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.

A Small Organic Molecule Blocks EGFR Transport into the Nucleus by the Nonclassical Pathway Resulting in Repression of Cancer Invasion.

In addition to the traditional epidermal growth factor receptor (EGFR) signaling pathways, nuclear EGFR has been shown to control multiple cellular functions, including cell proliferation and invasion. It has been reported that EGFR is transported into the nucleus after forming a complex with KPNA/KPNB1 or KPNB1. Herein, it is shown that EGFR can interact with both KP and KPNA, but EGF-activated EGFR mostly binds with KPNB1 through the pull-down assay. Also, a small organic molecule (1), an effective binder of KPNB1, inhibits the interaction between EGFR and KPNB1 in the nonclassical transport pathway, but not KPNA. Furthermore, treatment of cancer cells with 1 noticeably blocks the nuclear entry of EGFR, which results in significant suppression of invasion by lung cancer H1299 cells. These findings show that 1 is an effective inhibitor of EGFR/KPNB1 interactions in vitro, it may be used in cellular studies as a tool to determine the role of nuclear EGFR, and it is a drug candidate.

Transcriptome analysis for UVB-induced phototoxicity in mouse retina.

Throughout life, the human eye is continuously exposed to sunlight and artificial lighting. Ambient light exposure can lead to visual impairment and transient or permanent blindness. To mimic benign light stress conditions, Mus musculus eyes were exposed to low-energy UVB radiation, ensuring no severe morphological changes in the retinal structure post-exposure. We performed RNA-seq analysis to reveal the early transcriptional changes and key molecular pathways involved before the activation of the canonical cell death pathway. RNA-seq analysis identified 537 genes that were differentially modulated, out of which 126 were clearly up regulated (>2-fold, P < .01) and 51 were significantly down regulated (<2-fold, P < .01) in response to UVB irradiation in the mouse retina. Gene ontology analysis revealed that UVB exposure affected pathways for cellular stress and signaling (eg, Creb3, Ddrgk1, Grin1, Map7, Uqcc2, Uqcrb), regulation of chromatin and gene expression (eg, Chd5, Jarid2, Kat6a, Smarcc2, Sumo1, Zfp84), transcription factors (eg, Asxl2, Atf7, Per1, Phox2a, Rxra), RNA processing, and neuronal genes (eg, B4gal2, Drd1, Grm5, Rnf40, Rnps1, Usp39, Wbp4). The differentially expressed genes from the RNA-seq analysis were validated by quantitative PCR. Both analyses yielded similar gene expression patterns. The genes and pathways identified here improve the understanding of early transcriptional responses to UVB irradiation. They may also help in elucidating the genes responsible for the inherent susceptibility of humans to UVB-induced retinal diseases.

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.

Sox4-mediated caldesmon expression facilitates differentiation of skeletal myoblasts.

Caldesmon (CaD), which was originally identified as an actin-regulatory protein, is involved in the regulation of diverse actin-related signaling processes, including cell migration and proliferation, in various cells. The cellular function of CaD has been studied primarily in the smooth muscle system; nothing is known about its function in skeletal muscle differentiation. In this study, we found that the expression of CaD gradually increased as differentiation of C2C12 myoblasts progressed. Silencing of CaD inhibited cell spreading and migration, resulting in a decrease in myoblast differentiation. Promoter analysis of the caldesmon gene (Cald1) and gel mobility shift assays identified Sox4 as a major trans-acting factor for the regulation of Cald1 expression during myoblast differentiation. Silencing of Sox4 decreased not only CaD protein synthesis but also myoblast fusion in C2C12 cells and myofibril formation in mouse embryonic muscle. Overexpression of CaD in Sox4-silenced C2C12 cells rescued the differentiation process. These results clearly demonstrate that CaD, regulated by Sox4 transcriptional activity, contributes to skeletal muscle differentiation.

Inhibition of discoidin domain receptor 2-mediated lung cancer cells progression by gold nanoparticle-aptamer-assisted delivery of peptides containing transmembrane-juxtamembrane 1/2 domain.

The delivery of biologically functional peptides into mammalian cells can be a direct and effective method for cancer therapy and treatment of other diseases. Discoidin domain receptor 2 (DDR2) is a collagen-induced receptor tyrosine kinase recently identified as a novel therapeutic target in lung cancer. In this study, we report that peptides containing the functional domain of DDR2 can be efficiently delivered into lung malignant cancer cells via a gold nanoparticle-DNA aptamer conjugate (AuNP-Apt)-based system. Peptide delivery resulted in the abrogation of DDR2 activation triggered by collagen. Moreover, the peptide delivered by the AuNP-Apt system inhibited cancer cell proliferation and invasion mediated by DDR2 activation. Thus, these results suggest that peptide loaded onto AuNP-Apt conjugates can be used for the development of peptide-based biomedical applications for the treatment of DDR2-positive cancer.

Inhibition of Ku70 acetylation by INHAT subunit SET/TAF-Iß regulates Ku70-mediated DNA damage response.

DNA double-strand breaks (DSBs) can cause either cell death or genomic instability. The Ku heterodimer Ku70/80 is required for the NHEJ (non-homologous end-joining) DNA DSB repair pathway. The INHAT (inhibitor of histone acetyltransferases) complex subunit, SET/TAF-Iß, can inhibit p300- and PCAF-mediated acetylation of both histone and p53, thereby repressing general transcription and that of p53 target genes. Here, we show that SET/TAF-Iß interacts with Ku70/80, and that this interaction inhibits CBP- and PCAF-mediated Ku70 acetylation in an INHAT domain-dependent manner. Notably, DNA damage by UV disrupted the interaction between SET/TAF-Iß and Ku70. Furthermore, we demonstrate that overexpressed SET/TAF-Iß inhibits recruitment of Ku70/80 to DNA damage sites. We propose that dysregulation of SET/TAF-Iß expression prevents repair of damaged DNA and also contributes to cellular proliferation. All together, our findings indicate that SET/TAF-Iß interacts with Ku70/80 in the nucleus and inhibits Ku70 acetylation. Upon DNA damage, SET/TAF-Iß dissociates from the Ku complex and releases Ku70/Ku80, which are then recruited to DNA DSB sites via the NHEJ DNA repair pathway.

The intracellular juxtamembrane domain of discoidin domain receptor 2 (DDR2) is essential for receptor activation and DDR2-mediated cancer progression.

Discoidin domain receptors (DDRs) are unusual receptor tyrosine kinases (RTKs) that are activated by fibrillar collagens instead of soluble growth factors. DDRs play an important role in various cellular functions and disease processes, including malignant progression. Compared to other RTKs, DDRs have relatively long juxtamembrane domains, which are believed to contribute to receptor function. Despite this possibility, the function and mechanism of the juxtamembrane domain of DDRs have not yet been fully elucidated. In this study, we found that the cytoplasmic juxtamembrane 2 (JM2) region of DDR2 contributed to receptor dimerization, which is critical for receptor activation in response to collagen stimulation. A collagen-binding assay showed that JM2 was required for efficient binding of collagen to the discoidin (DS) domain. Immunohistochemical analysis of DDR2 expression using a tissue microarray demonstrated that DDR2 was overexpressed in several carcinoma tissues, including bladder, testis, lung, kidney, prostate and stomach. In H1299 cells, inhibition of DDR2 activity by overexpressing the juxtamembrane domain containing JM2 suppressed collagen-induced colony formation, cell proliferation and invasion via the inhibition of matrix metalloproteinase-2 and matrix metalloproteinase-9. Taken together, our results suggest that JM2-mediated dimerization is likely to be essential for DDR2 activation and cancer progression. Thus, inhibition of DDR2 function using a JM2-containing peptide might be a useful strategy for the treatment of DDR2-positive cancers.

Swiprosin-1 modulates actin dynamics by regulating the F-actin accessibility to cofilin.

Membrane protrusions, like lamellipodia, and cell movement are dependent on actin dynamics, which are regulated by a variety of actin-binding proteins acting cooperatively to reorganize actin filaments. Here, we provide evidence that Swiprosin-1, a newly identified actin-binding protein, modulates lamellipodial dynamics by regulating the accessibility of F-actin to cofilin. Overexpression of Swiprosin-1 increased lamellipodia formation in B16F10 melanoma cells, whereas knockdown of Swiprosin-1 inhibited EGF-induced lamellipodia formation, and led to a loss of actin stress fibers at the leading edges of cells but not in the cell cortex. Swiprosin-1 strongly facilitated the formation of entangled or clustered F-actin, which remodeled the structural organization of actin filaments making them in accessible to cofilin. EGF-induced phosphorylation of Swiprosin-1 at Ser183, a phosphorylation site newly identified using mass spectrometry, effectively inhibited clustering of actin filaments and permitted cofilin access to F-actin, resulting in actin depolymerization. Cells over expressing a Swiprosin-1 phosphorylation-mimicking mutant or a phosphorylation-deficient mutant exhibited irregular membrane dynamics during the protrusion and retraction cycles of lamellipodia. Taken together, these findings suggest that dynamic exchange of Swiprosin-1 phosphorylation and dephosphorylation is a novel mechanism that regulates actin dynamics by modulating the pattern of cofilin activity at the leading edges of cells.

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].

EGCG-induced selective death of cancer cells through autophagy-dependent regulation of the p62-mediated antioxidant survival pathway.

The effects of EGCG on the selective death of cancer cells by modulating antioxidant pathways through autophagy were explored in various normal and cancer cells. EGCG positively regulated the p62-KEAP1-NRF2-HO-1 pathway in normal cells, while negatively regulating it in cancer cells, leading to selective apoptotic death of cancer cells. In EGCG-treated MRC5 cells (EGCG-MRC5), autophagic flux was blocked, which was accompanied by the formation of p62-positive aggregates. However, EGCG-treated HeLa cells (EGCG-HeLa) showed incomplete autophagic flux and no aggregate formation. The levels of P-ULK1 S556 and S758 increased in EGCG-MRC5 through AMPK-mTOR cooperative interaction. In contrast, EGCG treatment in HeLa cells led to AMPK-induced mTOR inactivation, resulting in abrogation of P-ULK1 S556 and S758 levels. AMPK knockout in EGCG-HeLa restored positive regulation of the p62-mediated pathway, which was accompanied by increased P-mTOR S2448 and P-ULK1 S758 levels. Knockdown of 67LR in EGCG-HeLa abolished AMPK activity but did not restore the p62-mediated pathway. Surprisingly, both AMPK knockout and 67LR knockdown in EGCG-HeLa markedly increased cell viability, despite differential regulation of the antioxidant enzyme HO-1. In conclusion, EGCG induces the selective death of cancer cells through the modulation of at least two autophagy-dependent and independent regulatory pathways: negative regulation involves the mTOR-ULK1 (S556 and S758)-p62-KEAP1-NRF2-HO-1 axis via AMPK activation, whereas positive regulation occurs through the 67LR-AMPK axis.