Knockdown HeLa Cells Research Articles

Cardiomyocyte-specific deletion of the mitochondrial transporter Abcb10 causes cardiac dysfunction via lysosomal-mediated ferroptosis.

Heart function is highly dependent on mitochondria, which not only produce energy but also regulate many cellular functions. Therefore, mitochondria are important therapeutic targets in heart failure. Abcb10 is a member of the ABC transporter superfamily located in the inner mitochondrial membrane and plays an important role in haemoglobin synthesis, biliverdin transport, antioxidant stress, and stabilization of the iron transporter mitoferrin-1. However, the mechanisms underlying the impairment of mitochondrial transporters in the heart remain poorly understood. Here, we generated mice with cardiomyocyte-specific loss of Abcb10. The Abcb10 knockouts exhibited progressive worsening of cardiac fibrosis, increased cardiovascular risk markers and mitochondrial structural abnormalities, suggesting that the pathology of heart failure is related to mitochondrial dysfunction. As the mitochondrial dysfunction was observed early but mildly, other factors were considered. We then observed increased Hif1α expression, decreased NAD synthase expression, and reduced NAD+ levels, leading to lysosomal dysfunction. Analysis of ABCB10 knockdown HeLa cells revealed accumulation of Fe2+ and lipid peroxides in lysosomes, leading to ferroptosis. Lipid peroxidation was suppressed by treatment with iron chelators, suggesting that lysosomal iron accumulation is involved in ferroptosis. We also observed that Abcb10 knockout cardiomyocytes exhibited increased ROS production, iron accumulation, and lysosomal hypertrophy. Our findings suggest that Abcb10 is required for the maintenance of cardiac function and reveal a novel pathophysiology of chronic heart failure related to lysosomal function and ferroptosis.

Upregulation of SOX9 promotes the self-renewal and tumorigenicity of cervical cancer through activating the Wnt/β-catenin signaling pathway.

Sry-box9 (SOX9) maintains stem cell properties and plays crucial roles in many cancers. However, whether SOX9 is correlated with cervical cancer cell stemness and its detailed mechanism remains obscure. We studied the relationship between SOX9 and prognosis of cervical cancer through public database, and SOX9 was related to poor prognosis of cervical cancer. Elevated SOX9 expression enhanced the self-renewal properties and promotes tumorigenicity in cervical cancer. Overexpression of SOX9 could promote the expression of stem cell-related factors in cervical cancer cells and xenografts. Meanwhile, overexpression of SOX9 could also enhance the expressions of FZD10, β-catenin, and c-Myc in cervical cancer cells and xenografts, while inhibiting the expression of DDK1. The activation of Wnt pathway by chir-99 021 raised the tumor spheroid ability of SOX9 knockdown HeLa cells. In addition, SOX9 could transcriptional inhibit DKK1 and activate FZD10 and MYC by binding to their promoters to affect the Wnt/β-catenin pathway. These results demonstrated SOX9 regulated the self-renewal and tumorigenicity of cervical cancer through Wnt/β-catenin pathway by directly transcriptional activation of FZD10, MYC and transcriptional inhibition of DKK1.

The role and mechanism of tumor metastasis-associated gene 1 in radiosensitivity of HeLa cells

Objective: To determine the effect of tumor metastasis-associated gene 1 (MTA1) on the sensitivity of HeLa cells to radiotherapy, and to clarify its molecular mechanism. Methods: The transcriptome differences between MTA1 knocked down Hela cells and control cells were analyzed, and the differentially expressed genes (DEGs) was used to perform Gene-Set Enrichment Analysis (GSEA) and Gene Ontology (GO) cluster analysis. Flow cytometry was used to detect apoptosis in MTA1-overexpressed HeLa cells and control cells before and after 10 Gy X-ray irradiation. Cloning formation assay and real-time cellular analysis (RTCA) were used to monitor the cell proliferation before and after 2 Gy X-ray irradiation. To dissect the underlying molecular mechanisms of MTA1 affecting the sensitivity of radiotherapy, the proteins encoded by the DEGs were selected to construct a protein-protein interaction network, the expression of γ-H2AX was detected by immunofluorescence assay, and the expression levels of γ-H2AX, β-CHK2, PARP and cleaved caspase 3 were measured by western blot. Results: By transcriptome sequencing analysis, we obtained 649 DEGs, of which 402 genes were up-regulated in MTA1 knockdown HeLa cells and 247 genes were down-regulated. GSEA results showed that DEGs associated with MTA1 were significantly enriched in cellular responses to DNA damage repair processes. The results of flow cytometry showed that the apoptosis rate of MTA1 over-expression group (15.67±0.81)% after 10 Gy X-ray irradiation was significantly lower than that of the control group [(40.27±2.73)%, P<0.001]. After 2 Gy X-ray irradiation, the proliferation capacity of HeLa cells overexpressing MTA1 was higher than that of control cells (P=0.024). The numbers of colon in MTA1 over-expression group before and after 2 Gy X-ray irradiation were (176±7) and (137±7) respectively, higher than (134±4) and (75±4) in control HeLa cells (P<0.05). The results of immunofluorescence assay showed that there was no significant expression of γ-H2AX in MTA1 overexpressed and control HeLa cells without X-ray irradiation. Western blot results showed that the expression level of β-CHK2 in MTA1-overexpressing HeLa cells (1.04±0.06) was higher than that in control HeLa cells (0.58±0.25, P=0.036) after 10 Gy X-ray irradiation. The expression levels of γ-H2AX, PARP, and cleaved caspase 3 were 0.52±0.13, 0.52±0.22, and 0.63±0.18, respectively, in HeLa cells overexpressing MTA1, which were lower than 0.87±0.06, 0.78±0.12 and 0.90±0.12 in control cells (P>0.05). Conclusions: This study showed that MTA1 is significantly associated with radiosensitivity in cervical cancer HeLa cells. MTA1 over-expression obviously reduces the sensitivity of cervical cancer cells to X-ray irradiation. Mechanism studies initially indicate that MTA1 reduces the radiosensitivity of cervical cancer cells by inhibiting cleaved caspase 3 to suppress apoptosis and increasing β-CHK2 to promote DNA repair.

HPV E7 oncogene expression impairs Rb function and confers CDK4/6 inhibitor resistance in cervical cancer.

e17504 Background: Malignant tumor proliferation is one of the important factors for poor prognosis. CDK4/6 inhibitors can delay tumor progression by inhibiting cell cycle and inducing apoptosis, so they are potentially effective broad-spectrum anti-tumor targeted drugs, but drug resistance limits their clinical applications. Our previous research found that the CDK4/6 inhibitor LEE011 has anti-tumor effects on cervical cancer C33A cells, but no anti-tumor activity against HeLa. The mechanism is not very clear, we speculate that it is related to HPV infection. In this research, our aim is to clarify the correlation between HPV and tumor drug sensitivity and explore the possible mechanisms. Methods: Two HPV positive human cervical cancer cell lines, SiHa and Caski were chosen to verify the sensitivity to LEE011 by cell morphology, cell cycle and apoptosis. The antitumor activity of CDK4/6 inhibitor LEE011 by E7 knockin C33A and E7 knock-down HeLa cell lines was assessed by in vitro clonogenic assay, flow cytometry, and target inhibition verified by immunoblotting. Effects of LEE011 and E7 knock-down combination in vivo were studied by xenograft tumor regrowth delay, and tissue section immunohistochemistry. Results: SiHa and Caski were insensitive to LEE011 antitumor activity. Enhancement of drugsensitivity was lost in cell lines with HPV positive. After down-regulation of E7, LEE011 treatment increased the inhibition cell proliferation and pro-apoptosis of HeLa. Mechanistically, the loss of E7-induced Rb inactivation and LEE011 inhibited Rb phosphorylation, leading to cell-cycle arrest. In vivo, LEE011 inhibited tumor regrowth, with sustained inhibition of cyclin D-CDK4/6-Rb-E2F1 activity in E7 knock-down HeLa. However, up-regulation of E7 can reduce the ability of LEE011 to inhibit cell proliferation and pro-apoptosis of C33A. In summary, our study signifies inhibiting the CDK4/6 pathway by LEE011 in combination with down-regulation of E7 as a promising therapeutic strategy to treat cervical cancer. Conclusions: These findings suggested that HPV can affect Rb function by expressing oncoprotein E7 leading to resistance to targeted drug therapy, which means that HPV may be a biomarkers of drug therapy efficacy.

Iron metabolism protein transferrin receptor 1 involves in cervical cancer progression by affecting gene expression and alternative splicing in HeLa cells.

Transferrin receptor 1 (TfR1), encoded by TFRC, is a key regulator of iron homeostasis and plays important roles in many diseases, including cancers. To decipher the underlying molecular functions of TfR1 based on its influence on transcriptome profile in cancer cells. In this study, we first identified the expression pattern and prognostic influence of TFRC in cervical cancer patients from TCGA database. To explore the regulatory outcomes of TfR1 from the view of whole transcriptome profile, we generated TFRC knockdown (TFRC-KD) HeLa cells and negative control (NC) cells using short hairpin RNA (shRNA) method. Unbiased transcriptome sequencing (RNA-seq) experiment was used to analyze the global expression level and alternative splicing (AS) changes between TFRC-KD and NC cells. We found TFRC was consistently elevated in cervical cancer samples and tightly associated with prognosis of patients. Differential expression analysis revealed that 629 differentially expressed genes (DEGs) were identified between TFRC-KD and NC. Functional enrichment analysis of these DEGs revealed that TFRC-KD extensively disturbed cell physiology related pathways, including immunity, cell metabolism and gene expression. Moreover, dysregulated AS profile also indicated that TfR1 has important roles in the AS regulation. Hundreds of TfR1-regulated AS genes were involved in DNA repair, cell death, transcription and viral reproduction pathways, which were tightly associated with cancer cell progression. In summary, we for the first time explored the molecular functions of TfR1 at transcriptional and post-transcriptional levels. These results demonstrate TfR1 participates in the progression of cervical cancer by affecting the expression and AS levels of genes in cancer associated pathways, which greatly extends our understanding of TfR1 functions besides iron homeostasis and provide novel options in cancer treatment by targeting TfR1.

Knockdown of death receptor 5 antisense long noncoding RNA and cisplatin treatment modulate similar macromolecular and metabolic changes in HeLa cells.

Despite great progress in complex gene regulatory mechanisms in the dynamic tumor microenvironment, the potential contribution of long noncoding RNAs (lncRNAs) to cancer cell metabolism is poorly understood. Death receptor 5 antisense (DR5-AS) is a cisplatin inducible lncRNA whose knockdown modulates cell morphology. However, its effect on cell metabolism is unknown. The aim of this study is to examine metabolic changes modulated by cisplatin and DR5-AS lncRNA in HeLa cells. We used cisplatin as a universal cancer therapeutic drug to modulate metabolic changes in HeLa cervix cancer cells. We then examined the extent of metabolic changes by Fourier transform infrared spectroscopy (FTIR). We also performed transcriptomics analyses by generating new RNA-seq data with total RNAs isolated from cisplatin-treated HeLa cells. Then, we compared cisplatin-mediated transcriptomics and macromolecular changes with those mediated by DR5-AS knockdown. Cisplatin treatment caused changes in the unsaturated fatty acid and lipid-to-protein ratios and the glycogen content. These observations in altered cellular metabolism were supported by transcriptomics analyses. FTIR spectroscopy analyses have revealed that DR5-AS knockdown causes a 20.9% elevation in the lipid/protein ratio and a 76.6% decrease in lipid peroxidation. Furthermore, we detected a 3.42% increase in the chain length of the aliphatic lipids, a higher content of RNA, and a lower amount of glycogen indicating relatively lower metabolic activity in the DR5-AS knockdown HeLa cells. Interestingly, we observed a similar gene expression pattern under cisplatin treatment and DR5-AS knockdown HeLa cells. These results suggest that DR5-AS lncRNA appears to account for a fraction of cisplatin-mediated macromolecular and metabolic changes in HeLa cervix cancer cells.

Bclaf1 regulates c-FLIP expression and protects cells from TNF-induced apoptosis and tissue injury.

TNF stimulation generates pro-survival signals through activation of NF-κB that restrict the build-in death signaling triggered by TNF. The competition between TNF-induced survival and death signals ultimately determines the fate of a cell. Here, we report the identification of Bclaf1 as a novel component of the anti-apoptotic program of TNF. Bclaf1 depletion in multiple cells sensitizes cells to TNF-induced apoptosis but not to necroptosis. Bclaf1 exerts its anti-apoptotic function by promoting the transcription of CFLAR, a caspase 8 antagonist, downstream of NF-κB activation. Bclaf1 binds to the p50 subunit of NF-κB, which is required for Bclaf1 to stimulate CFLAR transcription. Finally, in Bclaf1 siRNA administered mice, TNF-induced small intestine injury is much more severe than in control mice with aggravated signs of apoptosis and pyroptosis. These results suggest Bclaf1 is a key regulator in TNF-induced apoptosis, both invitro and invivo.

Transcriptomics Profiling Identifies Cisplatin-Inducible Death Receptor 5 Antisense Long Non-coding RNA as a Modulator of Proliferation and Metastasis in HeLa Cells.

Cisplatin is a well-known cancer chemotherapeutic agent but how extensively long non-coding RNA (lncRNA) expression is modulated by cisplatin is unknown. It is imperative to employ a comprehensive approach to obtain a better account of cisplatin-mediated changes in the expression of lncRNAs. In this study, we used a transcriptomics approach to profile lncRNAs in cisplatin-treated HeLa cells, which resulted in identification of 10,214 differentially expressed lncRNAs, of which 2,500 were antisense lncRNAs. For functional analyses, we knocked down one of the cisplatin inducible lncRNAs, death receptor 5 antisense (DR5-AS) lncRNA, which resulted in a morphological change in HeLa cell shape without inducing any cell death. A second round of transcriptomics-based profiling revealed differential expression of genes associated with immune system, motility and cell cycle in DR5-AS knockdown HeLa cells. Cellular analyses showed that DR5-AS reduced cell proliferation and caused a cell cycle arrest at S and G2/M phases. Moreover, DR5-AS knockdown reduced the invasive capacity of HeLa cells in zebrafish xenograft model. These results suggest that cisplatin-mediated pleiotropic effects, such as reduction in cell proliferation, metastasis and cell cycle arrest, may be mediated by lncRNAs.

Crosstalk Between NDP52 and LUBAC in Innate Immune Responses, Cell Death, and Xenophagy.

Nuclear dot protein 52 kDa (NDP52, also known as CALCOCO2) functions as a selective autophagy receptor. The linear ubiquitin chain assembly complex (LUBAC) specifically generates the N-terminal Met1-linked linear ubiquitin chain, and regulates innate immune responses, such as nuclear factor-κB (NF-κB), interferon (IFN) antiviral, and apoptotic pathways. Although NDP52 and LUBAC cooperatively regulate bacterial invasion-induced xenophagy, their functional crosstalk remains enigmatic. Here we show that NDP52 suppresses canonical NF-κB signaling through the broad specificity of ubiquitin-binding at the C-terminal UBZ domain. Upon TNF-α-stimulation, NDP52 associates with LUBAC through the HOIP subunit, but does not disturb its ubiquitin ligase activity, and has a modest suppressive effect on NF-κB activation by functioning as a component of TNF-α receptor signaling complex I. NDP52 also regulates the TNF-α-induced apoptotic pathway, but not doxorubicin-induced intrinsic apoptosis. A chemical inhibitor of LUBAC (HOIPIN-8) cancelled the increased activation of the NF-κB and IFN antiviral pathways, and enhanced apoptosis in NDP52-knockout and -knockdown HeLa cells. Upon Salmonella-infection, colocalization of Salmonella, LC3, and linear ubiquitin was detected in parental HeLa cells to induce xenophagy. Treatment with HOIPIN-8 disturbed the colocalization and facilitated Salmonella expansion. In contrast, HOIPIN-8 showed little effect on the colocalization of LC3 and Salmonella in NDP52-knockout cells, suggesting that NDP52 is a weak regulator in LUBAC-mediated xenophagy. These results indicate that the crosstalk between NDP52 and LUBAC regulates innate immune responses, apoptosis, and xenophagy.

FKBP8 LIRL-dependent mitochondrial fragmentation facilitates mitophagy under stress conditions.

Mitochondrial quality control maintains mitochondrial function by regulating mitochondrial dynamics and mitophagy. Despite the identification of mitochondrial quality control factors, little is known about the crucial regulators coordinating both mitochondrial fission and mitophagy. Through a cell-based functional screening assay, FK506 binding protein 8 (FKBP8) was identified to target microtubule-associated protein 1 light chain 3 (LC3) to the mitochondria and to change mitochondrial morphology. Microscopy analysis revealed that the formation of tubular and enlarged mitochondria was observed in FKBP8 knockdown HeLa cells and the cortex of Fkbp8 heterozygote-knockout mouse embryos. Under iron depletion-induced stress, FKBP8 was recruited to the site of mitochondrial division through budding and colocalized with LC3. FKBP8 was also found to be required for mitochondrial fragmentation and mitophagy under hypoxic stress. Conversely, FKBP8 overexpression induced mitochondrial fragmentation in HeLa cells, human fibroblasts and mouse embryo fibroblasts (MEFs), and this fragmentation occurred in Drp1 knockout MEF cells, FIP200 knockout HeLa cells and BNIP3/NIX double knockout HeLa cells, but not in Opa1 knockout MEFs. Interestingly, we found an LIR motif-like sequence (LIRL), as well as an LIR motif, at the N-terminus of FKBP8 and LIRL was essential for both inducing mitochondrial fragmentation and binding of FKBP8 to OPA1. Together, we suggest that FKBP8 plays an essential role in mitochondrial fragmentation through LIRL during mitophagy and this activity of FKBP8 together with LIR is required for mitophagy under stress conditions.

A novel anti-cancer mechanism of Nutlin-3 through downregulation of Eag1 channel and PI3K/AKT pathway

Nutlin-3 shows a potent antitumor efficacy through downregulation of the cancerogenic ether à go-go 1 (Eag1) channel. However, the molecular mechanisms responsible for the regulation of Eag1 by Nutlin-3 in cancer cells remain unclear. In this study, we propose a novel anticancer mechanism of Nutlin-3, in which Nutlin-3 acts through the p53-Eag1-PI3K/AKT pathway. We first confirmed that Eag1 was downregulated through the activation of p53 by Nutlin-3. We then revealed that the inhibition of Eag1 electrophysiological function resulted in the decrease of viability, migration and invasion of HeLa cells. It is worth noting that the antitumor effect of Nutlin-3 was abolished in the Eag1 knockdown HeLa cell lines by siRNA. And Nutlin-3 can decrease the cell viability of H8 cells which were stably transfected with Eag1, but has no obvious inhibitory effect on blank H8 cells. Finally, we demonstrated that the decrease in Eag1 channel activity induced by Nutlin-3 treatment exerts anticancer activity by inhibiting the PI3K/AKT pathway. Our study therefore fills the gap between p53 pathway and its cellular function mediated by Eag1, shedding light on the new anti-cancer mechanism of Nutlin-3.

BubR1 Is Essential for Thio-Dimethylarsinic Acid-Induced Spindle Assembly Checkpoint and Mitotic Cell Death for Preventing the Accumulation of Abnormal Cells.

Thio-dimethylarsinic acid (thio-DMA) was detected in human urine after exposure to inorganic arsenic and arsenosugars consumed by marine algae. Our previous studies have shown that thio-DMA disturbed the cell cycle progression and arrested cells in mitosis, though the biological significance or the mechanism by which thio-DMA-induced mitotic phase accumulation occurs is yet to be understood. In this study, we showed that thio-DMA promotes the phosphorylation of BubR1 protein, which is one of the constituents of the spindle assembly checkpoint (SAC) complex and accumulates in the cell in mitotic phase. Binding of Mad2 to CDC20, also known as the marker of the mitotic checkpoint complex (MCC) formation during the activation of SAC, was enhanced and mitotic associated cell death by apoptosis was promoted in HeLa cells but not in HepG2 cells. Basal BubR1 protein level in HepG2 was 10-times lower than that of HeLa cells. Consequently, BubR1 knockdown HeLa cells were generated by small interfering RNA (siRNA) technique. The MCC formation and mitotic arrest induced by thio-DMA were completely inhibited in BubR1 knockdown cells. Moreover, BubR1 knockdown cells could survive in the medium containing higher concentrations of thio-DMA with some abnormalities such as larger cell size, huge nucleus, multiple nuclei, and abnormal DNA contents. Especially, cyclin B1 negative tetraploid cells, which signify interphase cells with tetraploid, increased and survived after 48-72 h treatment with thio-DMA. Thus, these results suggest that BubR1-mediated SAC activation and MCC formation are one of the defense systems for preventing the accumulation and survival of abnormal cells induced by thio-DMA.

DRG2 knockdown induces Golgi fragmentation via GSK3β phosphorylation and microtubule stabilization

The perinuclear stacks of the Golgi apparatus maintained by dynamic microtubules are essential for cell migration. Activation of Akt (protein kinase B, PKB) negatively regulates glycogen synthase kinase 3β (GSK3β)-mediated tau phosphorylation, which enhances tau binding to microtubules and microtubule stability. In this study, experiments were performed on developmentally regulated GTP-binding protein 2 (DRG2)-stably knockdown HeLa cells to determine whether knockdown of DRG2 in HeLa cells treated with epidermal growth factor (EGF) affects microtubule dynamics, perinuclear Golgi stacking, and cell migration. Here, we show that DRG2 plays a key role in regulating microtubule stability, perinuclear Golgi stack formation, and cell migration. DRG2 knockdown prolonged the EGF receptor (EGFR) localization in endosome, enhanced Akt activity and inhibitory phosphorylation of GSK3β. Tau, a target of GSK3β, was hypo-phosphorylated in DRG2-knockdown cells and showed greater association with microtubules, resulting in microtubule stabilization. DRG2-knockdown cells showed defects in microtubule growth and microtubule organizing centers (MTOC), Golgi fragmentation, and loss of directional cell migration. These results reveal a previously unappreciated role for DRG2 in the regulation of perinuclear Golgi stacking and cell migration via its effects on GSK3β phosphorylation, and microtubule stability.

Mechanism of the efflux transport of demethoxycurcumin-O-glucuronides in HeLa cells stably transfected with UDP-glucuronosyltransferase 1A1

Demethoxycurcumin (DMC) is a safe and natural food-coloring additive, as well as an agent with several therapeutic properties. However, extensive glucuronidation in vivo has resulted in its poor bioavailability. In this study, we aimed to investigate the formation of DMC-O-glucuronides by uridine 5'-diphospho-glucuronosyltransferase 1A1 (UGT1A1) and its transport by breast cancer resistance protein (BCRP) and multidrug resistance-associated proteins (MRPs) in HeLa cells stably transfected with UGT1A1 (named HeLa1A1 cells). The chemical inhibitors Ko143 (a selective BCRP inhibitor) and MK571 (a pan-MRP inhibitor) both induced an obvious decrease in the excretion rate of DMC-O-glucuronides and a significant increase in intracellular DMC-O-glucuronide concentrations. Furthermore, BCRP knock-down resulted in a marked reduction in the level of excreted DMC-O-glucuronides (maximal 55.6%), whereas MRP1 and MRP4 silencing significantly decreased the levels of excreted DMC-O-glucuronides (a maximum of 42.9% for MRP1 and a maximum of 29.9% for MRP3), respectively. In contrast, neither the levels of excreted DMC-O-glucuronides nor the accumulation of DMC-O-glucuronides were significantly altered in the MRP4 knock-down HeLa cells. The BCRP, MRP1 and MRP3 transporters were identified as the most important contributors to the excretion of DMC-O-glucuronides. These results may significantly contribute to improving our understanding of mechanisms underlying the cellular disposition of DMC via UGT-mediated metabolism.

Endoribonucleolytic Cleavage of m6A-Containing RNAs by RNase P/MRP Complex.

N6-methyladenosine (m6A) is the most abundant internal modification in RNAs and plays regulatory roles in a variety of biological and physiological processes. Despite its important roles, the molecular mechanism underlying m6A-mediated gene regulation is poorly understood. Here, we show that m6A-containing RNAs are subject to endoribonucleolytic cleavage via YTHDF2 (m6A reader protein), HRSP12 (adaptor protein), and RNase P/MRP (endoribonucleases). We demonstrate that HRSP12 functions as an adaptor to bridge YTHDF2 and RNase P/MRP, eliciting rapid degradation of YTHDF2-bound RNAs. Transcriptome-wide analyses show that m6A RNAs that are preferentially targeted for endoribonucleolytic cleavage have an HRSP12-binding site and a RNase P/MRP-directed cleavage site upstream and downstream of the YTHDF2-binding site, respectively. We also find that a subset of m6A-containing circular RNAs associates with YTHDF2 in an HRSP12-dependent manner and is selectively downregulated by RNase P/MRP. Thus, our data expand the known functions of RNase P/MRP to endoribonucleolytic cleavage of m6A RNAs.

O-GlcNAcylation regulates integrin-mediated cell adhesion and migration via formation of focal adhesion complexes

O-GlcNAcylation is a post-translational modification of a protein serine or threonine residue catalyzed by O-GlcNAc transferase (OGT) in the nucleus and cytoplasm. O-GlcNAcylation plays important roles in the cellular signaling that affect the different biological functions of cells, depending upon cell type. However, whether or not O-GlcNAcylation regulates cell adhesion and migration remains unclear. Here, we used the doxycycline-inducible short hairpin RNA (shRNA) system to establish an OGT knockdown (KD) HeLa cell line and found that O-GlcNAcylation is a key regulator for cell adhesion, migration, and focal adhesion (FA) complex formation. The expression levels of OGT and O-GlcNAcylation were remarkably suppressed 24 h after induction of doxycycline. Knockdown of OGT significantly promoted cell adhesion, but it suppressed the cell migration on fibronectin. The immunostaining with paxillin, a marker for FA plaque, clearly showed that the number of FAs was increased in the KD cells compared with that in the control cells. The O-GlcNAcylation levels of paxillin, talin, and focal adhesion kinase were down-regulated in KD cells. Interestingly, the complex formation between integrin β1, focal adhesion kinase, paxillin, and talin was greatly increased in KD cells. Consistently, levels of active integrin β1 were significantly enhanced in KD cells, whereas they were decreased in cells overexpressing OGT. The data suggest a novel regulatory mechanism for O-GlcNAcylation during FA complex formation, which thereby affects integrin activation and integrin-mediated functions such as cell adhesion and migration.

Autophagy\u2013lysosome pathway alterations and alpha-synuclein up-regulation in the subtype of neuronal ceroid lipofuscinosis, CLN5 disease

Neuronal ceroid lipofuscinoses (NCLs) are a group of inherited neurodegenerative lysosomal storage disorders. CLN5 deficiency causes a subtype of NCL, referred to as CLN5 disease. CLN5 is a soluble lysosomal protein with an unclear function in the cell. Increased levels of the autophagy marker protein LC3-II have been reported in several subtypes of NCLs. In this report, we examine whether autophagy is altered in CLN5 disease. We found that the basal level of LC3-II was elevated in both CLN5 disease patient fibroblasts and CLN5-deficient HeLa cells. Further analysis using tandem fluorescent mRFP-GFP-LC3 showed the autophagy flux was increased. We found the alpha-synuclein (α-syn) gene SNCA was highly up-regulated in CLN5 disease patient fibroblasts. The aggregated form of α-syn is well known for its role in the pathogenicity of Parkinson’s disease. Higher α-syn protein levels confirmed the SNCA up-regulation in both patient cells and CLN5 knockdown HeLa cells. Furthermore, α-syn was localized to the vicinity of lysosomes in CLN5 deficient cells, indicating it may have a lysosome-related function. Intriguingly, knocking down SNCA reversed lysosomal perinuclear clustering caused by CLN5 deficiency. These results suggest α-syn may affect lysosomal clustering in non-neuronal cells, similar to its role in presynaptic vesicles in neurons.

P23 protects the human aryl hydrocarbon receptor from degradation via a heat shock protein 90-independent mechanism

The aryl hydrocarbon receptor (AHR) is a ligand-activated signaling molecule which is involved in diverse biological functions ranging from cancer metastasis to immune regulation. This receptor forms a cytoplasmic complex with Hsp90, p23, and XAP2. We have previously reported that down-regulation of p23 triggers degradation of the AHR protein, uncovering a potentially dynamic event which controls the cellular AHR levels without ligand treatment. Here we investigate the underlying mechanisms for this p23 effect using wild-type HeLa and the p23 knockdown HeLa cells. Reduction of the Hsp90 and XAP2 contents, however, did not affect the AHR protein levels, implying that this p23 effect on AHR is more than just alteration of the cytoplasmic complex dynamics. Association of p23 with Hsp90 is not important for the modulation of the AHR levels since exogenous expression of p23 mutants with modest Hsp90-binding affinity effectively restored the AHR message and protein levels. The protein folding property of p23 which resides at the terminal 50-amino acid region is not involved for this p23 effect. Results from our interaction study using the affinity purified thioredoxin fusion proteins and GST fusion proteins showed that p23 directly interacts with AHR and the interaction surface lies within AHR amino acid 1–216 and p23 amino acid 1–110. Down-regulation of the p23 protein content promotes the ubiquitination of AHR, indicating that p23 protects AHR from the ubiquitin-meditated protein degradation.

Targeted Exome Sequencing of Krebs Cycle Genes Reveals Candidate Cancer-Predisposing Mutations in Pheochromocytomas and Paragangliomas.

Purpose: Mutations in Krebs cycle genes are frequently found in patients with pheochromocytomas/paragangliomas. Disruption of SDH, FH or MDH2 enzymatic activities lead to accumulation of specific metabolites, which give rise to epigenetic changes in the genome that cause a characteristic hypermethylated phenotype. Tumors showing this phenotype, but no alterations in the known predisposing genes, could harbor mutations in other Krebs cycle genes.Experimental Design: We used downregulation and methylation of RBP1, as a marker of a hypermethylation phenotype, to select eleven pheochromocytomas and paragangliomas for targeted exome sequencing of a panel of Krebs cycle-related genes. Methylation profiling, metabolite assessment and additional analyses were also performed in selected cases.Results: One of the 11 tumors was found to carry a known cancer-predisposing somatic mutation in IDH1 A variant in GOT2, c.357A>T, found in a patient with multiple tumors, was associated with higher tumor mRNA and protein expression levels, increased GOT2 enzymatic activity in lymphoblastic cells, and altered metabolite ratios both in tumors and in GOT2 knockdown HeLa cells transfected with the variant. Array methylation-based analysis uncovered a somatic epigenetic mutation in SDHC in a patient with multiple pheochromocytomas and a gastrointestinal stromal tumor. Finally, a truncating germline IDH3B mutation was found in a patient with a single paraganglioma showing an altered α-ketoglutarate/isocitrate ratio.Conclusions: This study further attests to the relevance of the Krebs cycle in the development of PCC and PGL, and points to a potential role of other metabolic enzymes involved in metabolite exchange between mitochondria and cytosol. Clin Cancer Res; 23(20); 6315-24. ©2017 AACR.

PTEN Signaling at the Tumor: Immune Interface

The phosphatase and tensin homolog, PTEN, modulates a wide array of signaling processes that control cell metabolism and proliferation. Preclinical studies in gliomas and prostate carcinomas found that loss of the Pten gene imparts a senescent phenotype with diminished response to radiation and suppressed anti-tumor immunity. We utilized two tumor models of Pten mutation and analyzed immunologically relevant phenotypic changes, including after administration of radiation. Cell cycle distribution was analyzed on HeLa cells with PTEN knockdown following a single fraction of 10 Gy radiation. To further assess radiation responsiveness, Pten wild type and knockout mouse TSA breast cancer cells were treated with 5 Gy of radiation and evaluated for colony formation. HMGB1 release was measured as a surrogate of immunogenic cell death: specifically, TSA cells expressing a recombinant HMGB1-RFA fusion protein were transfected with Pten siRNA and treated with a single fraction of radiation, after which secreted levels of the fusion protein were quantified. Additionally, Pten+/+ and Pten-/- TSA cells were treated with a single fraction of radiation, and PD-L1 expression was analyzed by qPCR 24 hours later. Cell cycle analysis revealed a defect of the DNA damage checkpoint in PTEN knockdown HeLa cells. Following radiation, 64% of control HeLa cells were arrested at G2/M compared to only 34% of PTEN knockdown cells. Furthermore, irradiated Pten-/- TSA cells showed more resilient colony formation than Pten+/+ cells. The analysis of HMGB1 as a surrogate of immunogenic cell death demonstrated a 27% relative decrease of secreted HMGB1 following a single fraction of high dose radiation in Pten-/- cells, as compared to the Pten+/+ population (P < 0.0001). In addition, gene expression analysis of Pten+/+ and Pten-/- TSA cells revealed a 2-fold baseline increase in PD-L1 expression in Pten knockout cells. PTEN loss is a frequent alteration in cancer. We found that PTEN deficiency in tumor cells results in a reduced checkpoint response and more resilient colony formation following radiation, suggesting increased resistance. Furthermore, decreased secretion of HMGB1 and increased gene expression of PD-L1 in the Pten-/- cells are predictive of an immune resistant phenotype. Given these features, PTEN deficient tumor cells may be more prone to survive radiation therapy, even with an accumulating DNA damage. This trait, together with their expression of PD-L1, may suggest utility in employing anti PD-L1 immunotherapy. Further characterization of the impact of PTEN mutation on tumor responses to radiation and immunotherapy will help guide future treatment strategies in patients with associated mutations.