Enzyme-independent role of EZH2 in regulating cell cycle progression via the SKP2-KIP/CIP pathway.

While EZH2 enzymatic activity is well-known, emerging evidence suggests that EZH2 can exert functions in a methyltransferase-independent manner. In this study, we have uncovered a novel mechanism by which EZH2 positively regulates the expression of SKP2, a critical protein involved in cell cycle progression. We demonstrate that depletion of EZH2 significantly reduces SKP2 protein levels in several cell types, while treatment with EPZ-6438, an EZH2 enzymatic inhibitor, has no effect on SKP2 protein levels. Consistently, EZH2 depletion leads to cell cycle arrest, accompanied by elevated expression of CIP/KIP family proteins, including p21, p27, and p57, whereas EPZ-6438 treatment does not modulate their levels. We also provide evidence that EZH2 knockdown, but not enzymatic inhibition, suppresses SKP2 mRNA expression, underscoring the transcriptional regulation of SKP2 by EZH2 in a methyltransferase-independent manner. Supporting this, analysis of the Cancer Genome Atlas database reveals a close association between EZH2 and SKP2 expression in human malignancies. Moreover, EZH2 depletion but not enzymatic inhibition positively regulates the expression of major epithelial-mesenchymal transition (EMT) regulators, such as ZEB1 and SNAIL1, in transformed cells. Our findings shed light on a novel mechanism by which EZH2 exerts regulatory effects on cell proliferation and differentiation through its methyltransferase-independent function, specifically by modulating SKP2 expression.

AhR signaling modulates Ferroptosis by regulating SLC7A11 expression.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that is pivotal in development, metabolic homeostasis, and immune responses. While recent research has highlighted AhR's significant role in modulating oxidative stress responses, its mechanistic relationship with ferroptosis-an iron-dependent, non-apoptotic cell death-remains to be fully elucidated. In our study, we discovered that AhR plays a crucial role in ferroptosis, in part by transcriptionally regulating the expression of the solute carrier family 7 member 11 (SLC7A11). Our findings indicate that both pharmacological inactivation and genetic ablation of AhR markedly enhance erastin-induced ferroptosis. This enhancement is achieved by suppressing SLC7A11, leading to increased lipid peroxidation. We also obtained evidence of post-translational modifications of SLC7A11 during ferroptosis. Additionally, we observed that indole 3-pyruvate (I3P), an endogenous ligand of AhR, protects cells from ferroptosis through an AhR-dependent mechanism. Based on these insights, we propose that AhR transcriptionally regulates the expression of SLC family genes, which in turn play a pivotal role in mediating ferroptosis. This underscores AhR's essential role in suppressing lipid oxidation and ensuring cell survival under oxidative stress.

MicroRNA-Gene Interactions Impacted by Toxic Metal(oid)s during EMT and Carcinogenesis.

Chronic environmental exposure to toxic metal(loid)s significantly contributes to human cancer development and progression. It is estimated that approximately 90% of cancer deaths are a result of metastasis of malignant cells, which is initiated by epithelial-mesenchymal transition (EMT) during early carcinogenesis. EMT is regulated by many families of genes and microRNAs (miRNAs) that control signaling pathways for cell survival, death, and/or differentiation. Recent mechanistic studies have shown that toxic metal(loid)s alter the expression of miRNAs responsible for regulating the expression of genes involved in EMT. Altered miRNA expressions have the potential to be biomarkers for predicting survival and responses to treatment in cancers. Significantly, miRNAs can be developed as therapeutic targets for cancer patients in the clinic. In this mini review, we summarize key findings from recent studies that highlight chemical-miRNA-gene interactions leading to the perturbation of EMT after exposure to toxic metal(loid)s including arsenic, cadmium, nickel, and chromium.

Oxidative stress modulates expression of immune checkpoint genes via activation of AhR signaling.

Reactive oxygen species (ROS) are by-products of metabolism of oxygen and they play an important role in normal homeostasis and cell signaling, as well as in the initiation of diseases including cancer when their production is upregulated. Thus, it is imperative to understand the cellular and molecular basis by which ROS impact on various biological and pathological processes. In this report, we show that human keratinocyte cell line (HaCaT) treated with hydrogen peroxide displayed an increased activity of AhR, leading to enhanced expression of its downstream targets including cytochrome P450 genes. Intriguingly, preincubation of the complete culture medium with hydrogen peroxide accelerated AhR activation and its downstream signaling. Subsequent mass spectrometric analysis reveals that the oxidant elicits the production of oxindole, a tryptophan catabolic product. We further demonstrate that 2-oxindole (a major form of oxindole) is capable of activating AhR, strongly suggesting that ROS may exert a significant impact on AhR signaling. Consistent with this, we also observe that hexavalent chromium [Cr(VI)], a heavy metal known to generate ROS in vivo, enhances AhR protein levels, as well as stimulates expression of CYP1A2 in an AhR-dependent manner. Significantly, we show that hydrogen peroxide and 2-oxindole induce expression of IDO1 and PD-L1, two immune checkpoint proteins. Given the role of IDO1 and PD-L1 in mediating T cell activity and/or differentiation, we postulate that ROS in the tumor microenvironment may play a crucial role in immune suppression via perturbing AhR signaling.

CBX8 interacts with chromatin PTEN and is involved in regulating mitotic progression.

OBJECTIVES: Besides its role in regulating phosphatidylinositol-3 kinase (PI3K) signalling in the cytosol, PTEN also has a nuclear function. In this study, we attempted to understand the mechanism of chromatin PTEN in suppressing chromosomal instability during cell division. MATERIALS AND METHODS: Immunocoprecipitation, ectopic expression, and deletional analyses were used to identify the physical interaction between Chromobox Homolog protein 8 (CBX8) and PTEN, as well as the functional domain(s) of PTEN mediating the interaction. Cell synchronization followed by immunoblotting was employed to study cell cycle regulation of CBX8 and the functional interaction between chromatin PTEN and CBX8. Small interfering RNAs (siRNAs) were used to study the role of PTEN and CBX8 in modulating histone epigenetic markers during the cell cycle. RESULTS: Polycomb group (PcG) proteins including CBXs function to repress gene expression in a wide range of organisms including mammals. We recently showed that PTEN interacted with CBX8, a component of Polycomb Repressing Complex 1 (PRC1), and that CBX8 co-localized with PTEN in the nucleus. CBX8 levels were high, coinciding with its phosphorylation in mitosis. Phosphorylation of CBX8 was associated with monoubiquitinated PTEN and phosphorylated-BubR1 on chromatin. Moreover, CBX8 played an important role in cell proliferation and mitotic progression. Significantly, downregulation of either PTEN or CBX8 induced H3K27Me3 epigenetic marker in mitotic cells. CONCLUSION: CBX8 is a new component that physically interacts with chromatin PTEN, playing an important role in regulating mitotic progression.

Ras sumoylation in cell signaling and transformation.

Ras proteins are small GTPases that participate in multiple signal cascades, regulating crucial cellular processes including cell survival, proliferation, and differentiation. Mutations or deregulated activities of Ras are frequently the driving force for oncogenic transformation and tumorigenesis. Posttranslational modifications play a crucial role in mediating the stability, activity, or subcellular localization/trafficking of numerous cellular regulators including Ras proteins. A series of recent studies reveal that Ras proteins are also regulated by sumoylation. All three Ras protein isoforms (HRas, KRas, and NRas) are modified by SUMO3. The conserved lysine42 appears to be the primary site for mediating sumoylation. Expression of KRasV12/R42 mutants compromised the activation of the Raf/MEK/ERK signaling axis, leading to a reduced rate of cell migration and invasion in vitro in multiple cell lines. Moreover, treatment of transformed pancreatic cells with a SUMO E2 inhibitor blocks cell migration in a concentration-dependent manner, which is associated with a reduced level of both KRas sumoylation and expression of mesenchymal cell markers. Furthermore, mouse xenograft experiments reveal that expression of a SUMO-resistant mutant appears to suppress tumor development in vivo. Combined, these studies indicate that sumoylation functions as an important mechanism in mediating the roles of Ras in cell proliferation, differentiation, and malignant transformation and that the SUMO-modification system of Ras oncoproteins can be explored as a new druggable target for various human malignancies.

Identification of Radil as a Ras binding partner and putative activator.

Ras genes are among the most frequently mutated oncogenes in human malignancies. To date, there are no successful anticancer drugs in the clinic that target Ras proteins or their pathways. Therefore, it is imperative to identify and characterize new components that regulate Ras activity or mediate its downstream signaling. To this end, we used a combination of affinity-pulldown and mass spectrometry to search for proteins that are physically associated with KRas. One of the top hits was Radil, a gene product with a Ras-association domain. Radil is known to be a downstream effector of Rap1, inhibiting RhoA signaling to regulate cell adhesion and migration. We demonstrate that Radil interacted with all three isoforms of Ras including HRas, NRas, and KRas, although it exhibited the strongest interaction with KRas. Moreover, Radil interacts with GTP-bound Ras more efficiently, suggesting a possibility that Radil may be involved in Ras activation. Supporting this, ectopic expression of Radil led to transient activation of mitogen-activated protein kinase kinase and extracellular signal-regulated kinase; Radil knockdown resulted in weakened activation of Ras downstream signaling components, which was coupled with decreased cell proliferation and invasion, and reduced expression of mesenchymal cell markers. Moreover, Radil knockdown greatly reduced the number of adhesion foci and depolymerized actin filaments, molecular processes that facilitate cancer cell migration. Taken together, our present studies strongly suggest that Radil is an important player for regulating Ras signaling, cell adhesion, and the epithelial-mesenchymal transition and may provide new directions for Ras-related anticancer drug development.

K-Ras Lys-42 is crucial for its signaling, cell migration, and invasion.

Ras proteins participate in multiple signal cascades, regulating crucial cellular processes, including cell survival, proliferation, and differentiation. We have previously reported that Ras proteins are modified by sumoylation and that Lys-42 plays an important role in mediating the modification. In the current study, we further investigated the role of Lys-42 in regulating cellular activities of K-Ras. Inducible expression of K-RasV12 led to the activation of downstream components, including c-RAF, MEK1, and extracellular signal-regulated kinases (ERKs), whereas expression of K-RasV12/R42 mutant compromised the activation of the RAF/MEK/ERK signaling axis. Expression of K-RasV12/R42 also led to reduced phosphorylation of several other protein kinases, including c-Jun N-terminal kinase (JNK), Chk2, and focal adhesion kinase (FAK). Significantly, K-RasV12/R42 expression inhibited cellular migration and invasion in vitro in multiple cell lines, including transformed pancreatic cells. Given that K-Ras plays a crucial role in mediating oncogenesis in the pancreas, we treated transformed pancreatic cells of both BxPC-3 and MiaPaCa-2 with 2-D08, a small ubiquitin-like modifier (SUMO) E2 inhibitor. Treatment with the compound inhibited cell migration in a concentration-dependent manner, which was correlated with a reduced level of K-Ras sumoylation. Moreover, 2-D08 suppressed expression of ZEB1 (a mesenchymal cell marker) with concomitant induction of ZO-1 (an epithelial cell marker). Combined, our studies strongly suggest that posttranslational modification(s), including sumoylation mediated by Lys-42, plays a crucial role in K-Ras activities in vivo.

RAS GTPases are modified by SUMOylation.

RAS proteins are GTPases that participate in multiple signal cascades, regulating crucial cellular processes including cell survival, proliferation, differentiation, and autophagy. Mutations or deregulated activities of RAS are frequently the driving force for oncogenic transformation and tumorigenesis. Given the important roles of the small ubiquitin-related modifier (SUMO) pathway in controlling the stability, activity, or subcellular localization of key cellular regulators, we investigated here whether RAS proteins are posttranslationally modified (i.e. SUMOylated) by the SUMO pathway. We observed that all three RAS protein isoforms (HRAS, KRAS, and NRAS) were modified by the SUMO3 protein. SUMOylation of KRAS protein, either endogenous or ectopically expressed, was observed in multiple cell lines. The SUMO3 modification of KRAS proteins could be removed by SUMO1/sentrin-specific peptidase 1 (SENP1) and SENP2, but not by SENP6, indicating that RAS SUMOylation is a reversible process. A conserved residue in RAS, Lys-42, was a site that mediates SUMOylation. Results from biochemical and molecular studies indicated that the SUMO-E3 ligase PIASγ specifically interacts with RAS and promotes its SUMOylation. Moreover, SUMOylation of RAS appeared to be associated with its activation. In summary, our study reveals a new posttranslational modification for RAS proteins. Since we found that HRAS, KRAS, and NRAS can all be SUMOylated, we propose that SUMOylation might represent a mechanism by which RAS activities are controlled.

Intracellular ROS levels determine the apoptotic potential of keratinocyte by Quantum Dot via blockade of AKT Phosphorylation.

Quantum dots (QDs) have shown great potential for biomedical use in a broad range including diagnostic agents. However, the regulatory mechanism of dermal toxicity is poorly understood. In this study, we investigated how QDs-induced apoptosis is regulated in human keratinocytes. We also examined the effect of carboxylic acid-coated QDs (QD 565 and QD 655) on reactive oxygen species (ROS) production and apoptosis-related cellular signalling. The viability of keratinocyte was inhibited by two types of QDs in a concentration-dependent manner. QDs induce ROS production and blockade of AKT phosphorylation. Moreover, the cleavage of AKT-dependent pro-apoptotic proteins such as poly (ADP-ribose) polymerase, caspases-3 and caspases-9 was significantly increased. We also found that a decrease in cellular ROS level by ROS scavenger, N-acetylcysteine (NAC), resulting in the abolishment of QDs-induced AKT de-phosphorylation and cellular apoptosis. Interestingly, QD 655 had a more cytotoxic effect including oxidative stress and AKT-dependent apoptosis than QD 565. In addition, QD 655 had the cytotoxic potential in the human skin equivalent model (HSEM). These data show that QD-induced intracellular ROS levels may be an important parameter in QD-induced apoptosis. These findings from this study indicate that intracellular ROS levels might determine the apoptotic potential of keratinocyte by QD via blockade of AKT phosphorylation.

WWP2 is required for normal cell cycle progression.

WWP2 is a ubiquitin E3 ligase belonging to the Nedd4-like family. Given that WWP2 target proteins including PTEN that are crucial for regulating cell proliferation or suppressing tumorigenesis, we have asked whether WWP2 plays a role in controlling cell cycle progression. Here we report that WWP2 is necessary for normal cell cycle progression as its silencing significantly reduces the cell proliferation rate. We have identified that an isoform of WWP2 (WWP2-V4) is highly expressed in the M phase of the cell cycle. Silencing of WWP2 accelerates the turnover of cyclin E, which is accompanied by increased levels of phospho-histone H3 (p-H3) and cyclin B. Moreover, silencing of WWP2 results in compromised phosphorylation of Akt(S473), a residue whose phosphorylation is tightly associated with the activation of the kinase. Combined, these results strongly suggest that WWP2 is an important component in regulating the Akt signaling cascade, as well as cell cycle progression.

Cdh1, a substrate-recruiting component of anaphase-promoting complex/cyclosome (APC/C) ubiquitin E3 ligase, specifically interacts with phosphatase and tensin homolog (PTEN) and promotes its removal from chromatin.

A pool of PTEN localizes to the nucleus. However, the exact mechanism by which nuclear PTEN is regulated remains unclear. We have recently reported that Plk1 specifically phosphorylates PTEN on Ser-380 during mitosis. Here we report that PTEN also localized to chromatin and that chromatin PTEN was removed by a proteasome-dependent process during mitotic exit. Pulldown analysis revealed that Cdh1, but not Cdc20, was significantly associated with PTEN. Cdh1 interacted with PTEN via two separate domains, and their interaction was enhanced by MG132, a proteasome inhibitor. Cdh1 negatively controlled the stability of chromatin PTEN by polyubiquitination. Phosphorylation of PTEN on Ser-380 impaired its interaction with Cdh1, thus positively regulating PTEN stability on chromatin. Significantly, the PTEN interaction with Cdh1 was phosphatase-independent, and Cdh1 knockdown via RNAi led to significant accumulation of chromatin PTEN, delaying mitotic exit. Combined, our studies identify Cdh1 as an important regulator of nuclear/chromatin PTEN during mitosis.

Plk1 protein phosphorylates phosphatase and tensin homolog (PTEN) and regulates its mitotic activity during the cell cycle.

PTEN is a well known tumor suppressor through the negative regulation of the PI3K signaling pathway. Here we report that PTEN plays an important role in regulating mitotic timing, which is associated with increased PTEN phosphorylation in the C-terminal tail and its localization to chromatin. Pulldown analysis revealed that Plk1 physically interacted with PTEN. Biochemical studies showed that Plk1 phosphorylates PTEN in vitro in a concentration-dependent manner and that the phosphorylation was inhibited by Bi2635, a Plk1-specific inhibitor. Deletional and mutational analyses identified that Plk1 phosphorylated Ser-380, Thr-382, and Thr-383, but not Ser-385, a cluster of residues known to affect the PTEN stability. Interestingly, a combination of molecular and genetic analyses revealed that only Ser-380 was significantly phosphorylated in vivo and that Plk1 regulated the phosphorylation, which was associated with the accumulation of PTEN on chromatin. Moreover, expression of phospho-deficient mutant, but not wild-type PTEN, caused enhanced mitotic exit. Taken together, our studies identify Plk1 as an important regulator of PTEN during the cell cycle.

Chromatin PTEN is involved in DNA damage response partly through regulating Rad52 sumoylation.

A pool of PTEN localizes to the nucleus. However, the exact mechanism of action of nuclear PTEN remains poorly understood. We have investigated PTEN's role during DNA damage response. Here we report that PTEN undergoes chromatin translocation after DNA damage, and that its translocation is closely associated with its phosphorylation on S366/T370 but not on S380. Deletional analysis reveals that the C2 domain of PTEN is responsible for its nuclear translocation after exposure to genotoxin. Both casein kinase 2 and GSK3ß are involved in the phosphorylation of the S366/T370 epitope, as well as PTEN's association with chromatin after DNA damage. Significantly, PTEN specifically interacts with Rad52 and colocalizes with Rad52, as well as γH2AX, after genotoxic stress. Moreover, PTEN is involved in regulating Rad52 sumoylation. Combined, our studies strongly suggest that nuclear/chromatin PTEN mediates DNA damage repair through interacting with and modulating the activity of Rad52.

Analysis for the potential of polystyrene and TiO2 nanoparticles to induce skin irritation, phototoxicity, and sensitization.

The human skin equivalent model (HSEM) is well known as an attractive alternative model for evaluation of dermal toxicity. However, only limited data are available on the usefulness of a HSEM for nanotoxicity testing. This study was designed to investigate cutaneous toxicity of polystyrene and TiO2 nanoparticles using cultured keratinocytes, a HSEM, and an animal model. In addition, we also evaluated the skin sensitization potential of nanoparticles using a local lymph node assay with incorporation of BrdU. Findings from the present study indicate that polystyrene and TiO2 nanoparticles do not induce phototoxicity, acute cutaneous irritation, or skin sensitization. Results from evaluation of the HSEMs correspond well with those from animal models. Our findings suggest that the HSEM might be a useful alternative model for evaluation of dermal nanotoxicity.

Assessment of dermal toxicity of nanosilica using cultured keratinocytes, a human skin equivalent model and an in vivo model.

Assessments of skin irritation potentials are important aspects of the development of nanotechnology. Nanosilica is currently being widely used for commercial purposes, but little literature is available on its skin toxicity and irritation potential. This study was designed to determine whether nanosilica has the potential to cause acute cutaneous toxicity, using cultured HaCaT keratinocytes (CHK), a human skin equivalent model (HSEM), and invivo model. Nanosilica was characterized by scanning electron microscopy. We evaluated the cytotoxic effects of nanosilica on CHKs and the HSEM. In addition, we also investigated whether two commercially available nanosilicas with different sizes (7 and 10-20 nm) have different effects. To confirm invitro results, we evaluated the irritation potentials of nanosilicas on rabbit skin. Nanosilicas reduced the cell viabilities of CHKs in a dose-dependent manner. However, the HSEM revealed no irritation at 500 microg/ml of nanosilica. Furthermore, this result concurred with Draize skin irritation test findings. The present study data indicate that nanosilica does not cause acute cutaneous irritation. Furthermore, this study shows that the HSEM used provides more useful screening data than the conventional cell culture model on the relative toxicities of NPs.

Transcriptional activation of the human Klotho gene by epidermal growth factor in HEK293 cells; role of Egr-1.

Klotho is an antiaging gene involved in the suppression of several age-related phenotypes, but few studies have examined the mechanism underlying the regulation of human Klotho gene expression. In this study, we investigated the transcriptional regulation of the Klotho gene by epidermal growth factor (EGF) in HEK293 human embryonic kidney cells. By using serial deletion constructs of the promoter, we identified a proximal 45 bp (-90 to -45) region responsible for EGF-induced promoter activity. The Egr-1-binding motif is located within this region. Forced expression of Egr-1 stimulated Klotho gene promoter activity. A point mutation in the Egr-1-binding motif abrogated promoter inducibility by EGF or ectopic Egr-1 expression. Knockdown of Egr-1 by expression of small interfering RNA (siRNA) attenuated EGF-induced Klotho promoter activity. Further analysis showed that the Ras/MEK/Erk signaling cascade is involved in EGF-induced activation of the Klotho promoter. We conclude that the Klotho gene is activated by EGF in HEK293 cells.