Sp1 Degradation Research Articles

Sex-dependent phosphorylation of Argonaute 2 reduces the mitochondrial translocation of miR-181c and induces cardioprotection in females

Obesity-induced cardiac dysfunction is growing at an alarming rate, showing a dramatic increase in global prevalence. Mitochondrial translocation of miR-181c in cardiomyocytes results in excessive reactive oxygen species (ROS) production during obesity. ROS causes Sp1, a transcription factor for MICU1, to be degraded via post-translational modification. The subsequent decrease in MICU1 expression causes mitochondrial Ca2+ accumulation, ultimately leading to a propensity for heart failure. Herein, we hypothesized that phosphorylation of Argonaute 2 (AGO2) at Ser 387 (in human) or Ser 388 (in mouse) inhibits the translocation of miR-181c into the mitochondria by increasing the cytoplasmic stability of the RNA-induced silencing complex (RISC). Initially, estrogen offers cardioprotection in pre-menopausal females against the consequences of mitochondrial miR-181c upregulation by driving the phosphorylation of AGO2. Neonatal mouse ventricular myocytes (NMVM) treated with insulin showed an increase in pAGO2 levels and a decrease in mitochondrial miR-181c expression by increasing the binding affinity of AGO2-GW182 in the RISC. Thus, insulin treatment prevented excessive ROS production and mitochondrial Ca2+ accumulation. In human cardiomyocytes, we overexpressed miR-181c to mimic pathological conditions, such as obesity/diabetes. Treatment with estradiol (E2) for 48 h significantly lowered miR-181c entry into the mitochondria through increased pAGO2 levels. E2 treatment also normalized Sp1 degradation and MICU1 transcription that normally occurs in response to miR-181c overexpression. We then investigated these findings using an in vivo model, with age-matched male, female and ovariectomized (OVX) female mice. Consistent with the E2 treatment, we show that female hearts express higher levels of pAGO2 and thus, exhibit higher association of AGO2-GW182 in cytoplasmic RISC. This results in lower expression of mitochondrial miR-181c in female hearts compared to male or OVX groups. Further, female hearts had fewer consequences of mitochondrial miR-181c expression, such as lower Sp1 degradation and significantly decreased MICU1 transcriptional regulation. Taken together, this study highlights a potential therapeutic target for conditions such as obesity and diabetes, where miR-181c is upregulated. New and noteworthyIn this study, we show that the phosphorylation of Argonaute 2 (AGO2) stabilizes the RNA-induced silencing complex in the cytoplasm, preventing miR-181c entry into the mitochondria. Furthermore, we demonstrate that treatment with estradiol can inhibit the translocation of miR-181c into the mitochondria by phosphorylating AGO2. This ultimately eliminates the downstream consequences of miR-181c overexpression by mitigating excessive reactive oxygen species production and calcium entry into the mitochondria.

Sonic hedgehog restrains the ubiquitin-dependent degradation of SP1 to inhibit neuronal/glial senescence associated phenotypes in chemotherapy-induced peripheral neuropathy via the TRIM25-CXCL13 axis

IntroductionChemotherapy-induced peripheral neuropathy (CIPN) is a common complication that affects an increasing number of cancer survivors. However, the current treatment options for CIPN are limited. Paclitaxel (PTX) is a widely used chemotherapeutic drug that induces senescence in cancer cells. While previous studies have demonstrated that Sonic hedgehog (Shh) can counteract cellular dysfunction during aging, its role in CIPN remains unknown. ObjectivesHerein, the aim of this study was to investigate whether Shh activation could inhibits neuronal/glial senescence and alleviates CIPN. MethodsWe treated ND7/23 neuronal cells and RSC96 Schwann cells with two selective Shh activators (purmorphamine [PUR] and smoothened agonist [SAG]) in the presence of PTX. Additionally, we utilized a CIPN mouse model induced by PTX injection. To assess cellular senescence, we performed a senescence-associated β-galactosidase (SA-β-gal) assay, measured reactive oxygen species (ROS) levels, and examined the expression of P16, P21, and γH2AX. To understand the underlying mechanisms, we conducted ubiquitin assays, LC-MS/MS, H&E staining, and assessed protein expression through Western blotting and immunofluorescence staining. ResultsIn vitro, we observed that Shh activation significantly alleviated the senescence-related decline in multiple functions included SA-β-gal activity, expression of P16 and P21, cell viability, and ROS accumulation in DRG sensory neurons and Schwann cells after PTX exposure. Furthermore, our in vivo experiments demonstrated that Shh activation significantly reduced axonal degeneration, demyelination, and improved nerve conduction. Mechanistically, we discovered that PTX reduced the protein level of SP1, which was ubiquitinated by the E3 ligase TRIM25 at the lysine 694 (K694), leading to increased CXCL13 expression, and we found that Shh activation inhibited PTX-induced neuronal/glial senescence and CIPN through the TRIM25-SP1-CXCL13 axis. ConclusionThese findings provide evidence for the role of PTX-induced senescence in DRG sensory neurons and Schwann cells, suggesting that Shh could be a potential therapeutic target for CIPN.

Construction of a multiple ligation-driven exponentially symmetric T7-transcription machinery for single-molecule monitoring of diverse single-nucleotide polymorphisms in human cancers

Single nucleotide polymorphism (SNP) is one of the most abundant genetic variations among individuals. Since one human disease is generally associated with several SNPs, simultaneous monitoring of diverse SNPs is particularly important. Herein, we demonstrate the construction of a multiple ligation-driven exponentially symmetric T7-transcription machinery for single-molecule monitoring of diverse SNPs. In presence of KRAS-135C and BRAF-V600E, ligation probes (i.e., LP-1 and LP-2) and template probes (i.e., TP-1-TP-2 duplex) are splinted together to construct transcription templates 1 and 2, and their corresponding T7 promoters will activate symmetric transcription amplifications to produce two reporter probes (i.e., RP-1 and RP-2). RP-1 and RP-2 selectively hybridize with signal probes (i.e., SP-1 and SP-2) to initiate cyclic degradation of SP-1 and SP-2 for liberating Cy3 and Cy5 as well as RP-1 and RP-2. Subsequently, RP-1 and RP-2 act as the ligation templates to splint LP-1/LP-2 and TP-1-TP-2 together, initiating new cycles of ligation-transcription-degradation for exponential accumulation of Cy3 and Cy5. KRAS-135C and BRAF-V600E mutants can be quantitatively detected by simply counting Cy3 and Cy5 molecules. This nanomachine exhibits a limit of detection (LOD) of 7.24 × 10−20 M for KRAS-135C and 3.72 × 10−20 M for BRAF-V600E with a dynamic range of 9 orders of magnitude. Moreover, it can differentiate SNPs with sequence homology, distinguish rare SNPs with 0.01 % mutation level, discriminate SNPs in cancer cells and normal cells, and even quantify SNPs in various cancer cells at single-cell level, providing a universal platform for genomic SNP-related biomedical studies and molecular diagnostics.

Abstract 18791: Sex Differences in the Phosphorylation of Argonaute 2 Result in Reduced Translocation of Micro RNA-181c Into the Mitochondria and Cardioprotection in Pre-Menopausal Females

Introduction: MicroRNAs (miRNAs) play a crucial role in regulating gene expression and have been implicated in various aspects of cardiovascular health and disease. miR-181c is known to be upregulated in obesity, and its translocation into cardiomyocyte mitochondria leads to excessive production of Reactive Oxygen Species(ROS). ROS-mediated degradation of Sp1, a transcription factor for MICU1, results indecreased MICU1 expression and subsequent mitochondrial [Ca2+] accumulation, leading to heart failure. We hypothesized that phosphorylation of Argonaute 2 (pAGO2) at Ser 387could inhibit the translocation of miR-181c into mitochondria by enhancing the stability of the RNA-induced silencing complex in the cytoplasm. Estrogen (E2) could provide cardioprotection in pre-menopausal women against the consequences of mitochondrial miR-181c upregulation by phosphorylating AGO2. Hypothesis: We hypothesize that sex differences in pAGO2 levels lead to reduced translocation of miR-181c into mitochondria, thereby influencing the cardiac response observed in pre-menopausal females compared to males. Methods: This study utilized male, female, and ovariectomized (OVX) mice as in vivo model. AC16 cardiomyocyte cell line was treated with E2 and transfected with miR-181c to mimic conditions of miR-181c upregulation. qRT-PCR was performed to assess miR-181c, SP1, and MICU1 expression levels. Western blot analysis was employed to detect protein levels. Results: Our results revealed significantly higher levels of pAGO2 in pre-menopausal female mice compared to males. This increased pAGO2 phosphorylation was associated with decreased translocation of miR-181c into mitochondria in pre-menopausal females, and this condition was reversed in OVX mice. In AC16 cells, E2 treatment normalized the aberrant degradation of Sp1 and the dysregulation of MICU1 induced by miR-181c overexpression. Conclusion: This study provides evidence of sex differences in pAGO2 levels, suggesting that reduced translocation of miR-181c into mitochondria contributes to the observed cardiac effects in pre-menopausal females.

N-terminal domain of classical swine fever virus Npro induces proteasomal degradation of specificity protein 1 with reduced HDAC1 expression to evade from innate immune responses.

Of the flaviviruses, only CSFV and bovine viral diarrhea virus express Npro as the non-structural protein which is not essential for viral replication but functions to dampen host innate immunity. We have deciphered a novel mechanism with which CSFV uses to evade the host antiviral immunity by the N-terminal domain of its Npro to facilitate proteasomal degradation of Sp1 with subsequent reduction of HDAC1 and ISG15 expression. This is distinct from earlier findings involving Npro-mediated IRF3 degradation via the C-terminal domain. This study provides insights for further studies on how HDAC1 plays its role in antiviral immunity, and if and how other viral proteins, such as the core protein of CSFV, the nucleocapsid protein of porcine epidemic diarrhea virus, or even other coronaviruses, exert antiviral immune responses via the Sp1-HDAC1 axis. Such research may lead to a deeper understanding of viral immune evasion strategies as part of their pathogenetic mechanisms.

LINC00955 suppresses colorectal cancer growth by acting as a molecular scaffold of TRIM25 and Sp1 to Inhibit DNMT3B-mediated methylation of the PHIP promoter

BackgroundLong non-coding RNAs play an important role in the development of colorectal cancer (CRC), while many CRC-related lncRNAs have not yet been identified.MethodsThe relationship between the expression of LINC00955 (Long Intergenic Non-protein Coding RNA 955) and the prognosis of colorectal cancer patients was analyzed using the sequencing results of the TCGA database. LINC00955 expression levels were measured using qRT-PCR. The anti-proliferative activity of LINC00955 was evaluated using CRC cell lines in vitro and xenograft models in nude mice in vivo. The interaction of TRIM25-Sp1-DNMT3B-PHIP-CDK2 was analyzed by western blotting, protein degradation experiment, luciferase, RNA-IP, RNA pull-down assays and immunohistochemically analysis. The biological roles of LINC00955, tripartite motif containing 25 (TRIM25), Sp1 transcription factor (Sp1), DNA methyltransferase 3 beta (DNMT3B), pleckstrin homology domain interacting protein (PHIP), cyclin dependent kinase 2 (CDK2) in colorectal cancer cells were analyzed using ATP assays, Soft agar experiments and EdU assays.ResultsThe present study showed that LINC00955 is downregulated in CRC tissues, and such downregulation is associated with poor prognosis of CRC patients. We found that LINC00955 can inhibit CRC cell growth both in vitro and in vivo. Evaluation of its mechanism of action showed that LINC00955 acts as a scaffold molecule that directly promotes the binding of TRIM25 to Sp1, and promotes ubiquitination and degradation of Sp1, thereby attenuating transcription and expression of DNMT3B. DNMT3B inhibition results in hypomethylation of the PHIP promoter, in turn increasing PHIP transcription and promoting ubiquitination and degradation of CDK2, ultimately leading to G0/G1 growth arrest and inhibition of CRC cell growth.ConclusionsThese findings indicate that downregulation of LINC00955 in CRC cells promotes tumor growth through the TRIM25/Sp1/DNMT3B/PHIP/CDK2 regulatory axis, suggesting that LINC00955 may be a potential target for the therapy of CRC.

The downregulation of HSP90-controlled CRALBP expression is associated with age-related vision attenuation.

The dysfunction of CRALBP, a key regulator of the visual cycle, is associated with retinitis punctata albescens characterized by night vision loss and retinal degeneration. In this paper, we find that the expression of CRALBP is regulated by heat shock protein 90 (HSP90). Inhibition of HSP90α or HSP90β expression by using the CRISPR-Cas9 technology downregulates CRALBP's mRNA and protein expression in ARPE-19 cells by triggering the degradation of transcription factor SP1 in the ubiquitin-proteasome pathway. SP1 can bind to CRALBP's promoter, and inhibition of SP1 by its inhibitor plicamycin or siRNA downregulates CRALBP's mRNA expression. In the zebrafish, inhibition of HSP90 by the intraperitoneal injection of IPI504 reduces the thickness of the retinal outer nuclear layer and Rlbp1b mRNA expression. Interestingly, the expression of HSP90, SP1, and CRALBP is correlatedly downregulated in the senescent ARPE-19 and Pig primary RPE cells in vitro and in the aged zebrafish and mouse retinal tissues in vivo. The aged mice exhibit the low night adaption activity. Taken together, these data indicate that the HSP90-SP1 is a novel regulatory axis of CRALBP transcriptional expression in RPE cells. The age-mediated downregulation of the HSP90-SP1-CRALBP axis is a potential etiology for the night vision reduction in senior people.

Transcription factor Sp1 regulates mitotic chromosome assembly and segregation

Aneuploidy is a pervasive feature of cancer cells that results from chromosome missegregation. Several transcription factors have been associated with aneuploidy; however, no studies to date have demonstrated that mammalian transcription factors directly regulate chromosome segregation during mitosis. Here, we demonstrate that the ubiquitously expressed transcription factor specificity protein 1 (Sp1), which we have previously linked to aneuploidy, has a mitosis-specific role regulating chromosome segregation. We find that Sp1 localizes to mitotic centromeres and auxin-induced rapid Sp1 degradation at mitotic onset results in chromosome segregation errors and aberrant mitotic progression. Furthermore, rapid Sp1 degradation results in anomalous mitotic chromosome assembly characterized by loss of condensin complex I localization to mitotic chromosomes and chromosome condensation defects. Consistent with these defects, Sp1 degradation results in reduced chromosome passenger complex activity and histone H3 serine 10 phosphorylation during mitosis, which is essential for condensin complex I recruitment and chromosome condensation. Together, these data provide the first evidence of a mammalian transcription factor acting specifically during mitosis to regulate chromosome segregation.

BAP1 phosphorylation-mediated Sp1 stabilization plays a critical role in cathepsin K inhibition-induced C-terminal p53-dependent Bax upregulation

Cathepsin K inhibitor (odanacatib; ODN) and cathepsin K knockdown (siRNA) enhance oxaliplatin-induced apoptosis through p53-dependent Bax upregulation. However, its underlying mechanisms remain unclear. In this study, we elucidated the mechanism behind enhancement of oxaliplatin-induced apoptosis by ODN. We also investigated the molecular mechanisms of ODN-induced Bax upregulation. Here, we demonstrated that ODN-induced Bax upregulation required p53, but it was independent of p53 transcriptional activity. Various mutants of the DNA-binding domain of p53 induced Bax upregulation in ODN-treated cells. p53 functional domain analysis showed that the C-terminal domain of p53 participates in the physical interaction and stabilization of Sp1, a major transcription factor of Bax. We screened a specific siRNA encoding 50 deubiquitinases and identified that BAP1 stabilizes Sp1. The knockdown or catalytic mutant form of BAP1 abolished the ODN-induced upregulation of Sp1 and Bax expression. Mechanistically, ODN induced BAP1 phosphorylation and enhanced Sp1-BAP1 interaction, resulting in Sp1 ubiquitination and degradation. Interestingly, ODN-induced BAP1 phosphorylation and DNA damage were modulated by the production of mitochondrial reactive oxygen species (ROS). Mitochondrial ROS scavengers prevented DNA damage, BAP1-mediated Sp1 stabilization, and Bax upregulation by ODN. BAP1 downregulation by siRNA inhibited apoptosis induced by the combined treatment of ODN and oxaliplatin/etoposide. Therefore, Sp1 is a crucial transcription factor for ODN-induced Bax upregulation, and Sp1 stabilization is regulated by BAP1.

Estradiol-mediated inhibition of Sp1 decreases miR-3194-5p expression to enhance CD44 expression during lung cancer progression

BackgroundSp1, an important transcription factor, is involved in the progression of various cancers. Our previous studies have indicated that Sp1 levels are increased in the early stage of lung cancer progression but decrease during the late stage, leading to poor prognosis. In addition, estrogen has been shown to be involved in lung cancer progression. According to previous studies, Sp1 can interact with the estrogen receptor (ER) to coregulate gene expression. The role of interaction between Sp1 and ER in lung cancer progression is still unknown and will be clarified in this study.MethodsThe clinical relevance between Sp1 levels and survival rates in young women with lung cancer was studied by immunohistochemistry. We validated the sex dependence of lung cancer progression in EGFRL858R-induced lung cancer mice. Wound healing assays, chamber assays and sphere formation assays in A549 cells, Taxol-induced drug-resistant A549 (A549-T24) and estradiol (E2)-treated A549 (E2-A549) cells were performed to investigate the roles of Taxol and E2 in lung cancer progression. Luciferase reporter assays, immunoblot and q-PCR were performed to evaluate the interaction between Sp1, microRNAs and CD44. Tail vein-injected xenograft experiments were performed to study lung metastasis. Samples obtained from lung cancer patients were used to study the mRNA level of CD44 by q-PCR and the protein levels of Sp1 and CD44 by immunoblot and immunohistochemistry.ResultsIn this study, we found that Sp1 expression was decreased in premenopausal women with late-stage lung cancer, resulting in a poor prognosis. Tumor formation was more substantial in female EGFRL858R mice than in male mice and ovariectomized female mice, indicating that E2 might be involved in the poor prognosis of lung cancer. We herein report that Sp1 negatively regulates metastasis and cancer stemness in E2-A549 and A549-T24 cells. Furthermore, E2 increases the mRNA and protein levels of RING finger protein 4 (RNF4), which is the E3-ligase of Sp1, and thereby decreases Sp1 levels by promoting Sp1 degradation. Sp1 can be recruited to the promoter of miR-3194-5p, and positively regulate its expression. Furthermore, there was a strong inverse correlation between Sp1 and CD44 levels in clinical lung cancer specimens. Sp1 inhibited CD44 expression by increasing the expression of miR-3194-5p, miR-218-5p, miR-193-5p, miR-182-5p and miR-135-5p, ultimately resulting in lung cancer malignancy.ConclusionPremenopausal women with lung cancer and decreased Sp1 levels have a poor prognosis. E2 increases RNF4 expression to repress Sp1 levels in premenopausal women with lung cancer, thus decreasing the expression of several miRNAs that can target CD44 and ultimately leading to cancer malignancy.

CREB/Sp1-mediated MCL1 expression and NFκB-mediated ABCB1 expression modulate the cytotoxicity of daunorubicin in chronic myeloid leukemia cells

Although some studies have hinted at the therapeutic potential of daunorubicin (DNR) in chronic myeloid leukemia (CML), the mechanism by which DNR induces CML cell death is unclear. Therefore, this study aimed to investigate DNR-induced cell death signaling pathways in CML cell lines K562 and KU812. DNR-triggered apoptosis in K562 cells was characterized by inhibition of MCL1 expression, while restoration of MCL1 expression protected K562 cells from DNR-mediated cytotoxicity. In addition, DNR induced NOX4-dependent ROS production, leading to the activation of p38 MAPK and inactivation of Akt and ERK. Activated p38 MAPK stimulated protein phosphatase 2A-dependent dephosphorylation of CREB. Since Akt-mediated activation of ERK reduced β-TrCP mRNA stability, the inactivation of Akt-ERK axis increased β-TrCP expression, which in turn promoted proteasomal degradation of Sp1. Inhibition of CREB phosphorylation and Sp1 expression simultaneously reduced MCL1 transcription and protein expression. DNR-induced MCL1 suppression was not reliant on its ability to induce DNA damage. In addition, DNR induced the expression of drug exporter ABCB1 in K562 cells through the p38 MAPK/NFκB-mediated pathway, while imatinib or ABT-199 inhibited the DNR-induced effect. The combination of imatinib or ABT-199 with DNR showed synergistic cytotoxicity in K562 cells by increasing intracellular DNR retention. Cumulatively, our data indicate that DNR induces MCL1 downregulation in K562 cells by promoting p38 MAPK-mediated dephosphorylation of CREB and inhibiting the Akt-ERK axis-mediated Sp1 protein stabilization. Furthermore, experimental evidence indicates that DNR-induced death of KU812 cells occurs through a similar pathway.

DNA damage-induced degradation of Sp1 promotes cellular senescence

Persistent DNA damage (genotoxic stress) triggers signaling cascades that drive cells into apoptosis or senescence to avoid replicating a damaged genome. Sp1 has been found to play a role in double strand break (DSB) repair, and a link between Sp1 and aging has also been established, where Sp1 protein, but not RNA, levels decrease with age. Interestingly, inhibition ATM reverses the age-related degradation of Sp1, suggesting that DNA damage signaling is involved in senescence-related degradation of Sp1. Proteasomal degradation of Sp1 in senescent cells is mediated via sumoylation, where sumoylation of Sp1 on lysine 16 is increased in senescent cells. Taking into consideration our previous findings that Sp1 is phosphorylated by ATM in response to DNA damage and that proteasomal degradation of Sp1 at DSBs is also mediated by its sumoylation and subsequent interaction with RNF4, we investigated the potential contribution of Sp1’s role as a DSB repair factor in mediating cellular senescence. We report here that Sp1 expression is decreased with a concomitant increase in senescence markers in response to DNA damage. Mutation of Sp1 at serine 101 to create an ATM phospho-null mutant, or mutation of lysine 16 to create a sumo-null mutant, prevents the sumoylation and subsequent proteasomal degradation of Sp1 and results in a decrease in senescence. Conversely, depletion of Sp1 or mutation of Sp1 to create an ATM phosphomimetic results in premature degradation of Sp1 and an increase in senescence markers. These data link a loss of genomic stability with senescence through the action of a DNA damage repair factor.

Inhibition of Sp1-mediated survivin and MCL1 expression cooperates with SLC35F2 and myeloperoxidase to modulate YM155 cytotoxicity to human leukemia cells

Although YM155 is reported to suppress survivin (also known as BIRC5) expression in cancer cells, its cytotoxic mechanism in human acute myeloid leukemia (AML) cells has not been clearly resolved. In this study, we analyzed the mechanistic pathways that modulate the sensitivity of human AML U937 and HL-60 cells to YM155. YM155 induced apoptosis in AML cells, which was characterized by p38 MAPK phosphorylation and downregulation of survivin and MCL1 expression. Phosphorylated p38 MAPK causes autophagy-mediated Sp1 degradation, thereby inhibiting the transcription of survivin and MCL1. The reduction of survivin and MCL1 levels further facilitated Sp1 protein degradation through autophagy. The restoration of Sp1, survivin, or MCL1 expression protected U937 and HL-60 cells from YM155-mediated cytotoxicity. U937 and HL-60 cells were continuously exposed to hydroquinone (HQ) to generate U937/HQ and HL-60/HQ cells, which showed increased SLC35F2 expression. The increase in SLC35F2 expression led to an increase in the sensitivity of U937/HQ cells to YM155-mediated cytotoxicity, whereas no such effect was observed in HL-60/HQ cells. Of note, myeloperoxidase (MPO) activity in HL-60 and HL-60/HQ cells enhanced YM155 cytotoxicity in these cells, and the enforced expression of MPO also increased the sensitivity of U937 cells to YM155. Taken together, we conclude that p38 MAPK-modulated autophagy inhibits Sp1-mediated survivin and MCL1 expression, which, in turn, leads to the death of U937 and HL-60 cells following YM155 treatment. In addition, our data indicate that SLC35F2 increases the sensitivity of U937 cells to YM155-mediated cytotoxicity, whereas MPO enhances YM155 cytotoxicity in U937 and HL-60 cells.

Sp1-independent downregulation of NHEJ in response to BER deficiency

Base excision repair (BER) is the major repair pathway that removes DNA single strand breaks (SSBs) arising spontaneously due to the inherent instability of DNA. Unrepaired SSBs promote cell-cycle delay, which facilitates DNA repair prior to replication. On the other hand, in response to persistent DNA strand breaks, ATM-dependent degradation of transcription factor Sp1 leads to downregulation of BER genes expression, further accumulation of SSBs and renders cells susceptible to elimination via apoptosis. In contrast, many cancer cells are not able to block replication and to downregulate the expression of Sp1 in response to DNA damage. However, knockdown of BER in cancer cells leads to the accumulation of DNA double strand breaks (DSBs), suggesting deficiency in non-homologous end joining (NHEJ) repair of DSBs. Here we investigated whether DNA repair deficiency caused by knockdown of the XRCC1 gene expression in proliferating cells results in downregulation of NHEJ genes expression. We find that knockdown of the XRCC1 gene expression does not cause degradation of Sp1, but leads to downregulation of Lig4/XRCC4 and Ku70/80 at the transcription and protein levels. We thus propose the existence of Sp1-independent backup mechanism that in response to BER deficiency downregulates NHEJ in proliferating cells.

Sp1 phosphorylation by ATM downregulates BER and promotes cell elimination in response to persistent DNA damage.

ATM (ataxia-telangiectasia mutated) is a central molecule for DNA quality control. Its activation by DNA damage promotes cell-cycle delay, which facilitates DNA repair prior to replication. On the other hand, persistent DNA damage has been implicated in ATM-dependent cell death via apoptosis; however, the mechanisms underlying this process remain elusive. Here we find that, in response to persistent DNA strand breaks, ATM phosphorylates transcription factor Sp1 and initiates its degradation. We show that Sp1 controls expression of the key base excision repair gene XRCC1, essential for DNA strand break repair. Therefore, degradation of Sp1 leads to a vicious cycle that involves suppression of DNA repair and further aggravation of the load of DNA damage. This activates transcription of pro-apoptotic genes and renders cells susceptible to elimination via both apoptosis and natural killer cells. These findings constitute a previously unrecognized ‘gatekeeper’ function of ATM as a detector of cells with persistent DNA damage.

Dihydroartemisinin induced caspase-dependent apoptosis through inhibiting the specificity protein 1 pathway in hepatocellular carcinoma SK-Hep-1 cells

AimsDihydroartemisinin (DHA) is a semi-synthetic derivative of artemisinin, well known for a safe and effective first-line antimalarial agent. This study investigated whether and how DHA induces apoptosis focusing on the specificity protein 1 (Sp1) pathway in hepatocellular carcinoma (HCC) SK-Hep-1 cells. Main methodsThe cell viability was evaluated by MTT assay. Cell cycle analysis was performed after PI staining by flow cytometry system. Apoptosis was confirmed by DAPI staining and by detecting cytoplasmic histone-associated-DNA-fragments using a cell death detection ELISAPLUS kit. The expression of proteins involved in apoptosis was evaluated by Western blot. The nuclear localization of Sp1 was evaluated by immunofluorescence assay. Key findingsDHA exerted potent cytotoxicity against HCC SK-Hep-1 cells compared with normal hepatocyte AML12 cells. The sub-G1 DNA content and apoptosis index were increased by DHA, which was accompanied by nuclei condensation and fragmentation. DHA activated caspase 3, caspase 8, and caspase 9 and cleaved poly (ADP-ribose) polymerase (PARP). DHA-induced apoptotic cell death, activation of caspases and cleavage of PARP were dramatically inhibited by pan caspase inhibitor Z-VAD-FMK. DHA down-regulated protein expression and nuclear localization of Sp1, which in turn decreased Sp1 downstream target protein, X-linked inhibitor of apoptosis. Decreased Sp1 protein expression by DHA was restored by proteasome inhibitor MG132. DHA led to a down-regulation of phospho-ERK, -p38 and -JNK without affecting their total forms. SignificanceThese results demonstrate that DHA induces caspase-dependent apoptosis in HCC SK-Hep-1 cells by proteasome-dependent degradation of Sp1, which is involved in mitogen-activate protein kinase pathway.

Quinacrine induces the apoptosis of human leukemia U937 cells through FOXP3/miR-183/β-TrCP/SP1 axis-mediated BAX upregulation

Quinacrine, which is clinically used as an antimalarial drug, has anti-cancer activity. However, mechanism underlying its cytotoxic effect remains to be completely elucidated. In the present study, we investigated the cytotoxic effect of quinacrine on human leukemia U937 cells. Quinacrine-induced apoptosis of U937 cells was accompanied with ROS generation, mitochondrial depolarization, and BAX upregulation. Quinacrine-treated U937 cells showed ROS-mediated p38 MAPK activation and ERK inactivation, which in turn upregulated FOXP3 transcription. FOXP3-mediated miR-183 expression decreased β-TrCP mRNA stability and suppressed β-TrCP-mediated SP1 degradation, thus increasing SP1 expression in U937 cells. Upregulated SP1 expression further increased BAX expression. BAX knock-down attenuated quinacrine-induced mitochondrial depolarization and increased the viability of quinacrine-treated cells. Together, our data indicate that quinacrine-induced apoptosis of U937 cells is mediated by mitochondrial alterations triggered by FOXP3/miR-183/β-TrCP/SP1 axis-mediated BAX upregulation.

Abstract 696: Inhibition of Ewing sarcoma cell growth by targeting Sp1 and survivin with the small molecule clotam

Abstract Ewing sarcoma (ES) is the most common soft tissue and bone tumor in adolescents and young adults. The survival rate of patients with metastatic disease is poor suggesting an urgent need for the development of more effective therapeutic options. Our aim was to investigate novel targets and identify less toxic agents to improve therapeutic efficacy in ES patients. Specificity protein 1 (Sp1) transcription factor regulates critical genes involved in cell proliferation. Sp1 also mediates the expression of survivin, an inhibitor of apoptosis protein. Overexpression of Sp1 and survivin is linked to aggressiveness and poor prognosis in several cancers; however, their precise association is not adequately evaluated in ES. Previously, we showed that tolfenamic acid (TA) inhibits leukemia, medulloblastoma and neuroblastoma cell proliferation by targeting Sp1. In this study, we investigated the anti-cancer activity of this small molecule using human ES cell lines. CHLA-9 and TC-32 cells were treated with vehicle (DMSO) or TA (2.5-20 µg/ml) and cell viability was measured at 24, 48 and 72 h post-treatment using CellTiter Glo kit. ES cells were treated with vehicle or TA (15 µg/ml) for 48 h. The mRNA and protein expression of Sp1 and survivin was determined by quantitative polymerase chain reaction (qPCR) and Western blot analysis respectively. Flow cytometry was used to measure apoptotic cell population using Annexin-V staining and cell cycle phase distribution with propidium iodide. Markers of apoptosis (caspase 3/7 activity using caspase Glo kit and cleaved PARP by Western blot) and cell cycle arrest (Cyclin D1 and p21 expression by Western blot) were also determined. Sp1 DNA-binding was evaluated by gel shift assay. TA treatment inhibited ES cell viability in a dose and time-dependent manner which is accompanied by the inhibition of Sp1 and survivin protein expression and a 2-3 fold increase in apoptotic markers, caspase 3/7 activity, Annexin-V staining, and c-PARP expression. Interestingly, TA inhibited mRNA expression of survivin but not Sp1, suggesting it has post-translational effects, perhaps proteasome-dependent degradation of Sp1. Gel shift assay revealed a decrease in Sp1 DNA-binding, indicating that TA could directly disrupt the binding of Sp1 with its consensus binding site in the promoter of target genes. Cyclin D1 is crucial for moving the cells from G0/G1 to next phases and induction of p21 is known to inhibit Cyclin D1. In this study, TA up-regulated p21, reduced Cyclin D1 expression and accumulated cells in G0/G1 phase. These results demonstrate that TA induces apoptosis and causes cell cycle arrest in ES cells by targeting Sp1 and survivin. Citation Format: Sagar Shelake, Umesh T. Sankpal, W. Paul Bowman, Matthew Wise, Anish Ray, Riyaz Basha. Inhibition of Ewing sarcoma cell growth by targeting Sp1 and survivin with the small molecule clotam [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 696. doi:10.1158/1538-7445.AM2017-696

ING4 suppresses tumor angiogenesis and functions as a prognostic marker in human colorectal cancer

ING4, a potential tumor suppressor, is implicated in cell cycle arrest, apoptosis, cell migration and angiogenesis. Here, we investigated the clinical value of ING4 and its impact on angiogenesis in colorectal cancer (CRC). In this study, we found that ING4 expression was significantly reduced in CRC tissues versus paired normal colon tissues. Moreover, low ING4 expression was significantly associated with increased lymph node metastasis, advanced TNM stage and poor overall survival. Multivariate Cox regression analysis showed that ING4 expression was an independent favourable prognostic factor for CRC (hazard ratio = 0.45, P = 0.001). In addition, we found that ING4 strongly inhibited CRC angiogenesis by suppressing Sp1 expression and transcriptional activity through ubiquitin degradation and down-regulating the expressions of Sp1 downstream pro-angiogenic genes, MMP-2 and COX-2. Moreover, ING4 might inhibit phosphorylation activity of cyclin/CDK2 complexes to trigger Sp1 degradation by inducing p21 expression in despite of p53 status. Our findings imply that reduced ING4 expression in CRC resulted in increased angiogenesis and contributed to CRC metastasis and poor prognosis. Restoration of ING4 may be a novel strategy for the treatment of metastatic CRC.

Synergistic targeting of Sp1, a critical transcription factor for myeloma cell growth and survival, by panobinostat and proteasome inhibitors.

Panobinostat, a pan-deacetylase inhibitor, synergistically elicits cytotoxic activity against myeloma (MM) cells in combination with the proteasome inhibitor bortezomib. Because precise mechanisms for panobinostat's anti-MM action still remain elusive, we aimed to clarify the mechanisms of anti-MM effects of panobinostat and its synergism with proteasome inhibitors. Although the transcription factor Sp1 was overexpressed in MM cells, the Sp1 inhibitor terameprocol induced MM cell death in parallel with reduction of IRF4 and cMyc. Panobinostat induced activation of caspase-8, which was inversely correlated with reduction of Sp1 protein levels in MM cells. The panobinostat-mediated effects were further potentiated to effectively induce MM cell death in combination with bortezomib or carfilzomib even at suboptimal concentrations as a single agent. Addition of the caspase-8 inhibitor z-IETD-FMK abolished the Sp1 reduction not only by panobinostat alone but also by its combination with bortezomib, suggesting caspase-8-mediated Sp1 degradation. The synergistic Sp1 reduction markedly suppressed Sp1-driven prosurvival factors, IRF4 and cMyc. Besides, the combinatory treatment reduced HDAC1, another Sp1 target, in MM cells, which may potentiate HDAC inhibition. Collectively, caspase-8-mediated post-translational Sp1 degradation appears to be among major mechanisms for synergistic anti-MM effects of panobinostat and proteasome inhibitors in combination.