Myeloid Cell Leukemia-1 Expression Research Articles

Oncolytic virotherapy promotes radiosensitivity in soft tissue sarcoma by suppressing anti-apoptotic MCL1 expression.

Soft tissue sarcoma (STS) is a rare cancer that develops from soft tissues in any part of the body. Despite major advances in the treatment of STS, patients are often refractory to conventional radiotherapy, leading to poor prognosis. Enhancement of sensitivity to radiotherapy would therefore improve the clinical outcome of STS patients. We previously revealed that the tumor-specific, replication-competent oncolytic adenovirus OBP-301 kills human sarcoma cells. In this study, we investigated the radiosensitizing effect of OBP-301 in human STS cells. The in vitro antitumor effect of OBP-301 and ionizing radiation in monotherapy or combination therapy was assessed using highly radiosensitive (RD-ES and SK-ES-1) and moderately radiosensitive (HT1080 and NMS-2) STS cell lines. The expression of markers for apoptosis and DNA damage were evaluated in STS cells after treatment. The therapeutic potential of combination therapy was further analyzed using SK-ES-1 and HT1080 cells in subcutaneous xenograft tumor models. The combination of OBP-301 and ionizing radiation showed a synergistic antitumor effect in all human STS cell lines tested, including those that show different radiosensitivity. OBP-301 was found to enhance irradiation-induced apoptosis and DNA damage via suppression of anti-apoptotic myeloid cell leukemia 1 (MCL1), which was expressed at higher levels in moderately radiosensitive cell lines. The combination of OBP-301 and ionizing radiation showed a more profound antitumor effect compared to monotherapy in SK-ES-1 (highly radiosensitive) and HT1080 (moderately radiosensitive) subcutaneous xenograft tumors. OBP-301 is a promising antitumor reagent to improve the therapeutic potential of radiotherapy by increasing radiation-induced apoptosis in STS.

MiR-26a Reverses Multidrug Resistance in Osteosarcoma by Targeting MCL1.

The multidrug resistance (MDR) acquired in human osteosarcoma is a huge obstacle for effective chemotherapy. Recently, microRNA-26a (miR-26a) has been associated with the pathogenesis and progression of osteosarcoma. However, whether it regulates MDR in osteosarcoma is unknown. We show here that miR-26a expression declines in chemoresistant osteosarcoma after neoadjuvant chemotherapy, and its expression correlates with clinical outcome. In addition, compared with sensitive parental cells, miR-26a expression also declines in osteosarcoma MDR cells, together suggesting a negative correlation between miR-26a expression and MDR development in osteosarcoma. We also show that the enforced expression of miR-26a reverses MDR in osteosarcoma cells, and conversely, miR-26a knockdown confers MDR in chemosensitive osteosarcoma cells treated with doxorubicin, methotrexate, or cisplatin. Mechanistically, miR-26a directly targets the pro-survival protein myeloid cell leukemia 1 (MCL1), and in turn, the enforced expression of MCL1 markedly antagonizes miR-26a-decreased MDR in osteosarcoma MDR cells, therefore demonstrating that miR-26a reverses MDR in osteosarcoma by targeting MCL1. Lastly, miR-26a reverses resistance to doxorubicin in osteosarcoma MDR cells xenografted in nude mice. Collectively, these results reveal a negative role and the underlying mechanism of miR-26a in the regulation of MDR in human osteosarcoma, implying a potential tactic of manipulating miR-26a for overcoming MDR in osteosarcoma chemotherapy.

Hsa_circ_0008537 facilitates liver carcinogenesis by upregulating MCL1 and Snail1 expression via miR‑153‑3p.

The biological functions of circular RNAs in liver tumorigenesis have been well demonstrated by a number of studies. Nevertheless, to the best of our knowledge, the role and mechanism of action of hsa_circ_0008537 (circ_0008537) in liver cancer pathogenesis remain undetermined. In the present study, circ_0008537 expression was associated with the GLI3 gene and was markedly increased in liver cancer tissue specimens and cells. High expression levels of circ_0008537 exhibited a poor prognosis. In addition, circ_0008537 overexpression resulted in an increased proliferation, migration and invasion of liver cancer cells, whereas circ_0008537 knockdown exhibited opposite effects. circ_0008537 acted as a sponge of microRNA-153-3p (miR-153-3p), and a negative correlation was observed between circ_0008537 and miR-153-3p expression in liver cancer. Transfection with miR-153-3p further abolished the effects of circ_0008537 on the malignant behavior of liver cancer cells. Furthermore, circ_0008537 indirectly affected the expression levels of pro-survival protein myeloid cell leukemia 1 (MCL1) and snail family zinc finger 1 (Snail1) via miR-153-3p in liver cancer cells. In conclusion, the data indicated that circ_0008537 facilitated liver carcinogenesis by indirectly regulating miR-153-3p and leading to the release of MCL1 and Snail1.

Long non-coding RNA SNHG20 promotes ovarian cancer development by targeting microRNA-338-3p to regulate MCL1 expression.

Long non-coding RNAs (lncRNAs) and microRNAs (miRNAs/miRs) were reported to be associated with the development of ovarian cancer (OC). Increasing evidence demonstrated that lncRNA SNHG20 and miR-338-3p were involved in OC. However, the functional mechanism of lncRNA SNHG20 and miR-338-3p in OC development remains unknown. The expression of SNHG20, miR-338-3p and myeloid cell leukemia 1 (MCL1) was detected by reverse transcription-quantitative PCR. MTT assay, flow cytometry and transwell migration and invasion assays were used to assess cell proliferation, apoptosis, migration and invasion, respectively. The relative protein expression was detected by western blot analysis. The interaction between miR-338-3p and SNHG20 or MCL1 was predicted by starBase v3.0, and subsequently confirmed by dual-luciferase reporter assay. Besides, mouse xenograft assay was carried out to explore the effect of SNHG20 on tumor growth in vivo. The levels of SNHG20 and MCL1 were upregulated, while miR-338-3p level was downregulated in OC tissues and cells. SNHG20 knockdown repressed OC cell proliferation, migration, invasion and epithelial-mesenchymal transition, and induced apoptosis. Interestingly, SNHG20 targeted miR-338-3p to regulate MCL1 expression. miR-338-3p depletion or MCL1 overexpression could reverse the effects of SNHG20 knockdown on OC cells. Besides, SNHG20 knockdown impeded tumor growth in vivo. In conclusion, the present study demonstrated that SNHG20 regulates OC development via modulation of the miR-338-3p/MCL1 axis, providing the theoretical basis for the treatment of OC.

A Novel BCL-2 Inhibitor APG-2575 Exerts Synthetic Lethality With BTK or MDM2-p53 Inhibitor in Diffuse Large B-Cell Lymphoma.

Despite therapeutic advances, the effective treatment for relapsed or refractory diffuse large B-cell lymphoma (DLBCL) remains a major clinical challenge. Evasion of apoptosis through upregulating antiapoptotic B-cell lymphoma-2 (BCL-2) family members and p53 inactivation, and abnormal activation of B-cell receptor signaling pathway are two important pathogenic factors for DLBCL. In this study, our aim is to explore a rational combination of BCL-2 inhibitor plus Bruton’s tyrosine kinase (BTK) blockade or p53 activation for treating DLBCL with the above characteristics. We demonstrated that a novel BCL-2 selective inhibitor APG-2575 effectively suppressed DLBCL with BCL-2 high expression via activating the mitochondrial apoptosis pathway. BTK inhibitor ibrutinib combined with BCL-2 inhibitors showed synergistic antitumor effect in DLBCL with mean expression of BCL-2 and myeloid cell leukemia-1 (MCL-1) through upregulating the expression level of BIM and modulating MCL-1 and p-Akt expression. For p53 wild-type DLBCL with high expression of BCL-2, APG-2575 showed strong synergic effect with mouse double minute 2 (MDM2)–p53 inhibitor APG-115 that can achieve potent antitumor effect and markedly prolong survival in animal models. Collectively, our data provide an effective and precise therapeutic strategy through rational combination of BCL-2 and BTK or MDM2–p53 inhibitors for DLBCL, which deserves further clinical investigation.

Intracellular levels of reactive oxygen species correlate with ABT‐263 sensitivity in non‐small‐cell lung cancer cells

ABT‐263 (Navitoclax) is a BH3‐mimetic drugs targeting anti‐apoptotic B‐cell lymphoma‐2 (BCL‐2) family proteins, including BCL‐2, BCL‐xL, and BCL‐w, thereby inducing apoptosis. In small‐cell lung cancer (SCLC) cells, the response to ABT‐263 is associated with the expression of myeloid cell leukemia‐1 (MCL‐1) protein, however the efficacy of ABT‐263 in non‐small‐cell lung cancer (NSCLC) has not been thoroughly evaluated. There are currently no established biomarkers for predicting the efficacy of ABT‐263 treatment in NSCLC. We screened a panel of different NSCLC cell lines and found that ABT‐263 inhibited cell proliferation and induced apoptosis in Calu‐1, Calu‐3, and BID007 cells. Inconsistent with previous reports on SCLC, low levels of MCL‐1 did not predict the response to ABT‐263 in NSCLC cells, however we found that intracellular levels of reactive oxygen species (ROS) in cancer cells were associated with sensitivity to ABT‐263 in NSCLC cells. We also showed that increasing the level of intracellular ROS could enhance the sensitivity to ABT‐263 in NSCLC cells. In summary, we propose that the intracellular levels of ROS could be used as a potential novel biomarker for predicting a response to ABT‐263 in NSCLC. Furthermore, we show some evidence supporting the further assessment of ABT‐263 as a new therapeutic strategy in patients with NSCLC combined with agents regulating ROS levels. We believe that our findings and follow‐up studies on this matter would lead to novel diagnostic and treatment strategies in patients with NSCLC.

Overexpression of Antiapoptotic MCL-1 Predicts Worse Overall Survival of Patients With Non-small Cell Lung Cancer

Myeloid cell leukemia-1 (MCL-1) is a member of the B-cell lymphoma-2 (Bcl-2) family of proteins, which regulate the intrinsic (mitochondrial) apoptotic cascade. MCL-1 inhibits apoptosis, which may be associated with resistance to cancer therapy. Therefore, in this study, the clinical role of MCL-1 in non-small cell lung cancer (NSCLC) was explored. This retrospective study included 80 patients with stage 1-3A NSCLC, who underwent surgery without preoperative treatment between 2010 and 2011. MCL-1 expression and Ki-67 index were determined via immunohistochemical staining. Apoptotic index (AI) was determined via terminal deoxynucleotidyl transferase dUTP nick end labeling. The receiver operating characteristic curve analysis (area under curve=0.6785) revealed that MCL-1 expression in 30.0% of the NSCLC tumor cells was a significant cut-off for predicting prognosis. Tumors were considered MCL-1-positive if staining was observed in >30% of the cells. Thirty-six tumors (45.0%) were MCL-1-positive. However, there were no significant differences between MCL-1 expression and clinical variables. AI was lower in MCL-1-positive (2.2±3.6%) than in MCL-1-negative (5.2±7.9%) tumors, although the difference was not significant (p=0.1080). The Ki-67 index was significantly higher in MCL-1-positive than in MCL-1-negative tumors (18.0% vs. 3.0%; p<0.001). Five-year survival rate was significantly worse in patients with MCL-1-positive tumors (68.3%) than in those with MCL-1-negative tumors (93.1%, p=0.0057). Univariate [hazard ratio (HR)=5.041, p=0.0013], and multivariate analyses revealed that MCL-1 expression was a significant prognostic factor (HR=3.983, p=0.0411). MCL-1 expression in NSCLC cells correlated inversely with AI and positively with Ki-67 index. MCL-1 may serve as a potential prognostic biomarker and a novel therapeutic target in NSCLC.

Expression of Myeloid Cell Leukemia-1 (Mcl-1) Predicts the Survival of Patients with Metastatic Gastric Cancer

Expression of Myeloid Cell Leukemia-1 (Mcl-1) Predicts the Survival of Patients with Metastatic Gastric Cancer

MCL1 Inhibitors in Multiple Myeloma

Apoptosis plays a key role, not only in normal homeostasis but also in protection against genomic instability. Protection against apoptosis is a hallmark of cancer and is mainly regulated by the overexpression of anti-apoptotic proteins such as Bcl-2, Bcl-Xl or Mcl-1. This results in increased survival of the tumor cells and resistance to therapy. This presentation will focus on MCL-1 (myeloid cell leukemia 1), its expression and its role as potential target in multiple myeloma (MM). MCL1 gene regions are one the most amplified gene regions in several human cancers and Mcl-1 activity is often associated with therapy resistance and relapse. Mcl-1 binds to and sequesters the pro-apoptotic BH3 proteins, thereby preventing apoptosis. Mcl-1 is overexpressed on MM cells from newly diagnosed patients compared to normal plasma cells and in MM cells at relapse. This overexpression is furthermore associated with a shorter survival of these patients. Increased Mcl-1 expression can result either from genetic lesions or by induction through interaction with the bone marrow microenvironment. Its expression is correlated with the molecular heterogeneity of the myeloma patients; while the CCDN1 group has high BCL2 and low MCL-1 expression; the MMSET and MAF group has high MCL-1 and low BCL2 expression. Unlike Bcl-2 and Bcl-Xl, Mcl-1 has a large unstructured aminoterminus and its activity is mainly dependent on posttranslational modifications. The bone marrow microenvironment, by producing high levels of interleukin 6, also induces the upregulation of Mcl-1. Furthermore, our group recently demonstrated that not only stromal cells in the bone marrow microenvironment, but also MDSC (myeloid derived suppressor cells) induce survival of MM cells by increasing Mcl-1 levels through the AMPK pathway. As such, these data suggest the potential therapeutic benefit of targeting Mcl-1 in MM patients. Developing the first-generation inhibitors appeared to be challenging, especially in view of the occurrence of unwanted off target effects. Recent preclinical data with new, selective Mcl-1 inhibitors show promising anti-tumor effects both in vitro and in in vivo myeloma models, either alone or in combination with the Bcl-2 selective inhibitor, venetoclax, especially as it was demonstrated that high levels of MCL-1 are associated with venetoclax resistance in MM. In addition, it was also shown that proteasome inhibition can trigger Mcl-1 accumulation, further pointing to the importance of Mcl-1 inhibition. Induction of NOXA, as an inhibitor of Mcl-1, is also suggested as a therapeutic option, especially in combinations with other drugs. Clinically, following preclinical results, several new Mcl-1 inhibitors have entered phase I trials. Most of them are still recruiting patients, and as such too early to have results. Disclosures No relevant conflicts of interest to declare.

MCL-1 Inhibition By the Selective MCL-1 Inhibitor AMG-176 Induces in Vitro Activity Against Burkitt Lymphoma Cell Lines and Synergistically Enhances the Cytotoxic Effect of Chemotherapy and BH3 Mimetics

Background: Although &gt;90% of Burkitt Lymphoma (BL) patients will be cured by intensive multi-agent immunochemotherapy, relapsed and refractory BL patients have poor prognoses demonstrating the need to identify novel therapies. Myeloid cell leukemia-1 (MCL-1) is a pro-survival protein within the BCL-2 family that has been investigated as a therapeutic target in several leukemia and lymphoma pre-clinical models and clinical studies. Analysis of pediatric BL tumors has implicated MCL-1 in BL pathogenesis (Giulino-Roth et al., Blood 2012) and targeting MCL-1 may be effective in MYC-driven lymphomas (Kelly et al., Genes Dev 2014). High MCL-1 expression has also been associated with therapy resistance and can confer resistance to BH3 mimetic agents (Carrington et al., Cell Death Differ 2017). AMG176, a selective MCL-1 inhibitor, is an effective promoter of apoptosis and is currently under investigation in clinical trials for relapsed and refractory multiple myeloma, lymphoma, and acute myeloid leukemia. Hypothesis: We hypothesized that MCL-1 inhibition with AMG176 will promote apoptosis and increase sensitivity of BL therapy-sensitive (TSCL) and therapy-resistant (TRCL) cell lines to other chemotherapy drugs. Methods: A panel of TSCLs (Raji, Ramos and Daudi) and one TRCL (Raji 4RH) BL cell lines were exposed to increasing concentrations of AMG176 as a single agent and in combination with Doxorubicin or Venetoclax at different time points. Cell viability was analyzed using PrestoBlue. Synergy in drug combination exposures was determined by Calcusyn software and reported as a combination index (CI) value with values increasingly &lt;1 indicating increasing synergistic activity. Apoptosis induction was analyzed by flow cytometry for Annexin V (AV) and Sytox-7AAD and by western blotting for PARP. Effects of MCL-1 inhibition on the expression of other pro- and anti-apoptotic BCL-2 family member proteins was assessed by western blotting. AMG176 mechanism(s) of action was evaluated by co-immunoprecipitation followed by western blotting for pro-apoptotic BCL-2 family members known to be bound by MCL-1. Results: Single agent AMG176 exposure of BL cell lines exhibited a dose and time-dependent decrease in viable cells. 48hr IC50 values of Ramos (0.3097uM) and Raji (1.652uM) showed an increased sensitivity to AMG176 as compared to Daudi (9.616uM) and Raji 4RH (12.45uM). Exposure of cells to AMG176 also resulted in altered expression levels of pro-survival and pro-apoptotic proteins of the BCL-2 family proteins. Co-immunoprecipitation performed after 48hrs of AMG176 exposure exhibited a decrease in MCL-1:BIM and MCL-1:BAK complexes in Raji and Ramos cells promoting a pro-apoptotic environment. After 48hr exposure to AMG176, induction of apoptosis was observed in a dose-dependent manner in all cell lines as evidenced by increased PARP cleavage on western blot and AV-Sytox positivity on flow cytometry. To investigate the ability of MCL-1 inhibition to enhance the activity of cytotoxic chemotherapy, BL cells were exposed to AMG176 in combination with Doxorubicin. Daudi and Raji exhibited strong synergy with peak synergistic activity observed at 72hrs (Daudi CI values &lt;0.1 at all AGM176 concentrations with 0.1-0.2uM Doxorubicin; Raji CI ranged from 0.233-0.295 at multiple AMG176 concentrations with &lt;0.4uM Doxorubicin) while a minimal effect was observed in Ramos and Raji 4RH. As high MCL-1 is associated with resistance to Venetoclax by sequestering free BIM following Venetoclax exposure, the combination of AMG176 and Venetoclax was investigated and also showed significant synergy in BL cells. A minimal effect was observed in Daudi and Raji 4RH in Venetoclax combination but Ramos and Raji, the more AMG176 sensitive cell lines, showed peak synergy at 24hrs at high doses of Venetoclax (Ramos CI values ranged from 0.3-0.478; Raji CI ranged from 0.511 to 0.67). Conclusion: MCL-1 inhibition with AMG176 demonstrates dose- and time-dependent anti-lymphoma activity impairing in vitro proliferation and inducing apoptosis in BL cells. AMG176 exhibits synergistic activity in combination with cytotoxic chemotherapy in both therapy-sensitive and therapy-resistant BL cell lines and synergistically enhances response to the BH3 mimetic agent Venetoclax. These findings highlight the potential of MCL-1 inhibition as a therapeutic option in BL. This work was supported by T35 NIH training grant (T35AI089693). Disclosures No relevant conflicts of interest to declare.

Cinobufagin, a bufadienolide from traditional Chinese medicine Bufo bufo gargarizans CANTOR, inhibits PC3 cell growth in vitro and in vivo

Abstract Objective To explore the effects of cinobufagin (CBF), an active component of toad venom (Bufo bufo gargarizans CANTOR), on the proliferation and apoptosis of PC3 human prostate cancer cells in vitro and preliminarily investigate the mechanism of CBF in suppressing tumor cell growth in vivo. Methods The effect of CBF on PC3 cells proliferation was detected using MTT assay. The morphological changes of PC3 cells were observed under an optical microscope. Colony formation assays were used to observe the CBF effect on colony formation by PC3 cells. PC3 cell apoptosis after treatment with CBF for 48 hours was monitored using flow cytometry. Furthermore, the effect of CBF on the expression of myeloid cell leukemia-1 (MCL-1) and related apoptotic proteins was examined using western blotting. A xenograft model was established in BALB/c nude mice to evaluate the effect of CBF on prostate cancer in vivo. Results The MTT assay results illustrated that PC3 cell proliferation was inhibited in vitro by CBF in a concentration- and time-dependent manner. Compared with the control group findings, CBF significantly inhibited the formation of PC3 cells (P = .005). Flow cytometry revealed that after treatment with 50 nM CBF for 48 hours, the apoptotic rate of PC3 cells was 41.97 (5.16)%, indicating that CBF could significantly induce its apoptosis (P = .003). In addition, optical and fluorescence microscopy uncovered remarkable inhibition of cell proliferation accompanied by morphologic changes. The western blotting result indicated that CBF obviously downregulated the expression of the anti-apoptotic protein MCL-1. Most importantly, CBF reduced the carcinogenicity of PC3 xenografts in nude mice. Conclusion CBF can inhibit the growth of PC3 cells both in vitro and in vivo and induce apoptosis of tumor cells. The corresponding mechanism may be correlated with the activation of caspase family proteins via MCL-1.

The regulatory protein GADD34 inhibits TRAIL-induced apoptosis via TRAF6/ERK-dependent stabilization of myeloid cell leukemia 1 in liver cancer cells

GADD34 (growth arrest and DNA damage-inducible gene 34) plays a critical role in responses to DNA damage and endoplasmic reticulum stress. GADD34 has opposing effects on different stimuli-induced cell apoptosis events, but the reason for this is unclear. Here, using immunoblotting analyses and various molecular genetic approaches in HepG2 and SMMC-7721 cells, we demonstrate that GADD34 protects hepatocellular carcinoma (HCC) cells from tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis by stabilizing a BCL-2 family member, myeloid cell leukemia 1 (MCL-1). We found that GADD34 knockdown decreased MCL-1 levels and that GADD34 overexpression up-regulated MCL-1 expression in HCC cells. GADD34 did not affect MCL-1 transcription but enhanced MCL-1 protein stability. The proteasome inhibitor MG132 abrogated GADD34 depletion-induced MCL-1 down-regulation, suggesting that GADD34 inhibits the proteasomal degradation of MCL-1. Furthermore, GADD34 overexpression promoted extracellular signal-regulated kinase (ERK) phosphorylation through a signaling axis that consists of the E3 ubiquitin ligase tumor necrosis factor receptor-associated factor 6 (TRAF6) and transforming growth factor-β-activated kinase 1 (MAP3K7)-binding protein 1 (TAB1), which mediated the up-regulation of MCL-1 by GADD34. Of note, TRAIL up-regulated both GADD34 and MCL-1 levels, and knockdown of GADD34 and TRAF6 suppressed the induction of MCL-1 by TRAIL. Correspondingly, GADD34 knockdown potentiated TRAIL-induced apoptosis, and MCL-1 overexpression rescued TRAIL-treated and GADD34-depleted HCC cells from cell death. Taken together, these findings suggest that GADD34 inhibits TRAIL-induced HCC cell apoptosis through TRAF6- and ERK-mediated stabilization of MCL-1.

MCL1 gene silencing promotes senescence and apoptosis of glioma cells via inhibition of the PI3K/Akt signaling pathway.

Glioma is known to be the most prevalent primary brain tumor. In recent years, there has been evidence indicating myeloid cell leukemia-1 (MCL1) plays a role in brain glioblastoma. Therefore, the present study was conducted with aims of exploring the ability of MCL1 silencing to influence glioma cell senescence and apoptosis through the mediation of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) signaling pathway. Glioma and tumor-adjacent tissues were collected in order to detect the presence of higher levels of MCL1 protein expression. Next, the mRNA and protein expression of MCL1, PI3K, Akt, B cell lymphoma 2 (Bcl2), Bcl2-associated X (Bax), B lymphoma Mo-MLV insertion region 1 homolog (Bmi-1), and phosphatase and tensin homolog (PTEN) were determined. Cell counting kit-8 assay was applied to detect cell proliferation, β-galactosidase staining for cell senescence, and flow cytometry for cell cycle entry and apoptosis. Initially, the results revealed higher positive expression rate of MCL1 protein, increased mRNA and protein expression of MCL1, PI3K, Akt, Bmi-1, and Bcl-2 and decreased that of Bax and PTEN in human glioma tissues. The silencing of MCL1 resulted in a decrease in mRNA and protein expression of PI3K, Akt, Bmi-1, and Bcl-2 and an increase in Bax and PTEN expressions in glioma cells. Moreover, silencing of MCL1 also inhibited cell proliferation and cell cycle entry in glioma cells, and promoted glioma cell senescence and apoptosis. In conclusion, the aforementioned results collectively suggested that the silencing of MCL1 promotes senescence and apoptosis in glioma cells through inhibiting the PI3K/Akt signaling pathway. Thus, decreasing the expression of MCL1 might have therapeutic functions in glioma. © 2018 IUBMB Life, 71(1):81-92, 2019.

To study the correlation of peripheral blood F-box and WD repeat domain-containing domain protein7 and myeloid cell leukemia-1 expression levels with the prognosis of Icotinib administration in elderly patients with advanced non-small-cell lung cancer

Objective To study the correlation of the prognosis of Icotinib administration with the expression levels of F-box and WD repeat domain-containing 7(FBW7) and myeloid cell leukemia-1(MCL-1) in peripheral blood in elderly patients with advanced non-small-cell lung cancer. Methods A total of 76 patients aged 60 years or over diagnosed with non-small-cell lung cancer(NSCLC)with EGFR-sensitive mutations and under Icotinib treatment were enrolled in this study.FBW7 and MCL-1 mRNA expression levels in peripheral blood were detected by real-time quantitative PCR(RT-QPCR). The correlation of FBW7 and MCL-1 expression levels with clinical and histological parameters, overall survival(OS), and progression-free-survival(PFS)was analyzed. Results The FBW7 expression level and the MCL-1 expression level were negative correlated(r=-0.37, P<0.001). High FBW7 expression levels and low MCL-1 expression levels in peripheral blood were associated with improved therapeutic efficacy of Icotinib (P<0.001) and extended OS and PFS.Cox regression analysis showed that the expression levels of FBW7 and MCL-1 in peripheral blood were independent influencing factors for OS and PFS. Conclusions Patients with high FBW7 expression levels and low MCl-1 expression levels are more likely to benefit from Icotinib treatment.Expression levels for either factor can be used as a predictive indicator for the effectiveness of Icotinib and provide guidance for its clinical use. Key words: Carcinoma, non-small-cell lung; Ubiquitin-protein ligases; Myeloid cell leukemia-1; Icotinib

Ursolic acid facilitates apoptosis in rheumatoid arthritis synovial fibroblasts by inducing SP1-mediated Noxa expression and proteasomal degradation of Mcl-1.

Rheumatoid arthritis (RA) is characterized by hyperplastic pannus formation mediated by activated synovial fibroblasts (RASFs) that cause joint destruction. We have shown earlier that RASFs exhibit resistance to apoptosis, primarily as a result of enhanced expression of myeloid cell leukemia-1 (Mcl-1). In this study, we discovered that ursolic acid (UA), a plant-derived pentacyclic triterpenoid, selectively induces B-cell lymphoma 2 homology 3-only protein Noxa in human RASFs. We observed that UA-induced Noxa expression was followed by a consequent decrease in Mcl-1 expression in a dose-dependent manner. Subsequent evaluation of the signaling pathways showed that UA-induced Noxa is primarily mediated by the JNK pathway in human RASFs. Chromatin immunoprecipitation (IP) studies into the promoter region of Noxa indicated the role of transcription factor specificity protein 1 in JNK-mediated Noxa expression. Furthermore, the results from IP studies and proximity ligation assays indicated that UA-induced Noxa colocalizes and associates with Mcl-1 to prime it for proteasomal degradation through K48-linked ubiquitination by the selective recruitment of Mcl-1 ubiquitin ligase E3, a homologous to E6-associated protein C terminus domain-containing E3 ubiquitin ligase. These findings unveil a novel mechanism of inducing apoptosis in RASFs and a potential adjunct therapeutic strategy of regulating synovial hyperplasia in RA.-Kim, E. Y., Sudini, K., Singh, A. K., Haque, M., Leaman, D., Khuder, S., Ahmed, S. Ursolic acid facilitates apoptosis in rheumatoid arthritis synovial fibroblasts by inducing SP1-mediated Noxa expression and proteasomal degradation of Mcl-1.

PTBP1 enhances miR-101-guided AGO2 targeting to MCL1 and promotes miR-101-induced apoptosis

Myeloid cell leukemia 1 (MCL1) is a key anti-apoptotic protein belonging to the BCL-2 protein family. To preserve normal cellular homeostasis, cells must maintain strict control over MCL1 expression. Overexpression of MCL1 has been identified as a key contributor to tumorigenesis, and further enables resistance to a number of anti-cancer chemotherapies. Thus, there is an ongoing interest to develop selective MCL1 inhibitors. In order to better target MCL1, it is essential to understand the molecular mechanisms that regulate MCL1 expression in cells. While MCL1 expression is tightly controlled by multiple mechanisms, the post-transcriptional regulation of MCL1 mRNA is poorly studied. Our previous work identified that polypyrimidine tract binding protein 1 (PTBP1) binds to MCL1 mRNA and represses MCL1 expression by destabilizing MCL1 mRNA. In this report, we show that PTBP1 modulates MCL1 expression by regulating the microRNA (miRNA) direction of the miRNA-induced silencing complex (miRISC) to MCL1. We demonstrate that PTBP1 enhances miR-101-guided AGO2 interaction with MCL1, thereby regulating miR-101-induced apoptosis and clonogenic cell survival inhibition in cells. Taken together, not only do these studies expand our understanding on the regulation of MCL1, they also demonstrate that PTBP1 and miRNAs can function cooperatively on a shared target mRNA.

Imperatorin Targets MCL-1 to Sensitize CD133+ Lung Cancer Cells to γδ-T Cell-Mediated Cytotoxicity

Background/Aims: CD133⁺ cancer cells display low sensitivity to anti-cancer treatment; thus, combination treatment with adjuvant drugs is required to improve the efficiency of cancer therapy. The aim of this study was to explore the effect of imperatorin, a linear furanocoumarin compound, on γδ T cell-mediated cytotoxicity against CD133⁺ lung cancer cells. Methods: CD133⁺ and CD133^- subgroups from A549 and PC9 lung cancer cells were sorted by using flow cytometry. The cytotoxicity of γδ T cells against cancer cells was evaluated by measuring lactate dehydrogenase release. The concentration of tumor necrosis factor-related apoptosis-inducing ligand in the co-culture system was determined by using an enzyme-linked immunosorbent assay. Mitochondrial membrane potential, expression of death receptor 4 (DR4) and DR5 on the cell surface, and rate of apoptosis were measured by flow cytometry. Cytochrome c release and cellular protein expression were detected by western blot analysis. Results: Compared with CD133^- cells, CD133⁺ cells were resistant to γδ T cell-mediated cytotoxicity. However, imperatorin significantly increased the sensitivity of CD133⁺ lung cancer cells to γδ T cell treatment in vitro and in vivo. Mechanically, we found that myeloid cell leukemia 1 (MCL-1), an important anti-apoptotic protein belonging to the Bcl-2 family, was overexpressed in CD133⁺ A549 and PC9 cells compared to their corresponding CD133^- cells. Co-treatment with imperatorin and γδ T cells suppressed the expression of MCL-1, and thus promoted the mitochondrial apoptosis mediated by γδ T cells in CD133⁺ A549 and PC9 lung cancer cells. Conclusion: Up-regulated MCL-1 in CD133⁺ lung cancer cells is responsible for their resistance to γδ T cells. Furthermore, the combination of γδ T cells with imperatorin sensitized CD133⁺ lung cancer cells to γδ T cell-mediated cytotoxicity by targeting MCL-1.

Abstract 4315: PTBP1 modulation of MCL1 mRNA regulates sensitivity to antitubulin chemotherapeutics

Abstract Background: Myeloid Cell Leukemia 1 (MCL1), an anti-apoptotic Bcl-2 family protein, is a key regulator of intrinsic apoptosis. Normal cells require strict control over MCL1 expression and aberrant MCL1 expression has been shown linked to the emergence of various diseases and chemoresistance. Previous studies have detailed how MCL1 expression is regulated by multiple mechanisms both transcriptionally and translationally. However, characterization of the post-transcriptional regulators of MCL1 mRNA is limited. Polypyrimidine tract binding protein 1 (PTBP1) is a known regulator of post-transcriptional gene expression that can control mRNA splicing, translation, stability, and localization. In the described study we characterize the impact that PTBP1 has on MCL1 expression, its molecular mechanisms, and its roles on cellular apoptosis. Methods: Cross-linking Immunoprecipitation (CLIP)-seq data analysis and RNA immunoprecipitation (RIP) were performed to analyze protein interactions with RNA and map the RNA binding sites on the genes of interests. RNA half-life was measured by treating cells with Actinomycin D and extracting total RNA at different time point. Cell viability was measured by the MTS assay and apoptosis was assessed by Annexin V/Propidium Iodide (PI) staining followed by flow cytometry analysis. Results: Our studies demonstrate that PTBP1 binds to MCL1 mRNA and that knockdown of PTBP1 up-regulates MCL1 expression in cancer. Mechanistically, PTBP1 silencing stabilizes MCL1 mRNA and increases MCL1 mRNA accumulation in cytoplasm. We further identify that this post-transcriptional regulation is mediated by microRNAs and Argonaute2 (Ago2). Moreover, we show that PTBP1 down-regulation is anti-apoptotic to antitubulin chemotherapeutics in a MCL1-dependent manner. Conclusions: Our findings suggest that PTBP1 is a novel post-transcriptional regulator of MCL1 mRNA by which it controls apoptotic response to antitubulin chemotherapeutics. Note: This abstract was not presented at the meeting. Citation Format: William J. Placzek, Jia Cui. PTBP1 modulation of MCL1 mRNA regulates sensitivity to antitubulin chemotherapeutics [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 4315. doi:10.1158/1538-7445.AM2017-4315

Enhanced Stabilization of MCL1 by the Human T-Cell Leukemia Virus Type 1 bZIP Factor Is Modulated by Blocking the Recruitment of Cullin 1 to the SCF Complex.

Human T-cell leukemia virus type 1 (HTLV-1) is an oncogenic retrovirus that is the etiological agent of adult T-cell leukemia (ATL). The HTLV-1 basic leucine zipper factor (HBZ), which is encoded by the minus strand of the provirus, is constitutively expressed in all ATL patient cells and likely contributes to the development and maintenance of ATL. Furthermore, the overexpression of the myeloid cell leukemia 1 (MCL1) protein is frequently observed in hematological cancers as well as several other types of cancers. Here, we found that the expression of HBZ in cells stabilized MCL1 protein expression and suppressed the MCL1-mediated release of cytochrome c from the mitochondria. This effect was mediated by inhibition of the ubiquitin-dependent degradation of MCL1. In a serial binding assay, HBZ interacted with cullin 1 (CUL1) through a head-to-tail interaction. The association between CUL1 and Skp1, which serves as the molecular scaffold for the components of SCF ubiquitin ligase complexes, was markedly repressed in the presence of HBZ. Mechanistic analysis indicated that HBZ abrogated the CUL1 association with Skp1, which in turn promoted the cellular expression of MCL1. This novel function of HBZ likely plays a role in the viral pathogenesis of HTLV-1 and provides important insights into our understanding of the development of ATL.

Downregulation of miR-29a/b/c in placenta accreta inhibits apoptosis of implantation site intermediate trophoblast cells by targeting MCL1

ObjectivePlacenta accreta is defined as abnormal adhesion of placental villi to the uterine myometrium. Although this condition has become more common as a result of the increasing rate of cesarean sections, the underlying causative mechanism(s) remain elusive. Because microRNA-29a/b/c (miR-29a/b/c) have been shown to play important roles in placental development, this study evaluated the roles of these microRNAs in placenta accreta. MethodsExpression of miR-29a/b/c and myeloid cell leukemia-1 (MCL1) were quantified in patient tissues and HTR8/SVneo trophoblast cells using the real-time quantitative polymerase chain reaction. Western blotting was used to analyze expression of the MCL1 protein in HTR8/SVneo trophoblast cells with altered expression of miR-29a/b/c. To determine their role in apoptosis, miR-29a/b/c were overexpressed in HTR-8/SVneo cells, and levels of apoptosis were analyzed by flow cytometry. Luciferase activity assays were used to determine whether MCL1 is a target gene of miR-29a/b/c. ResultsExpression of miR-29a/b/c was significantly lower in creta sites compared to noncreta sites (p = 0.018, 0.041, and 0.022, respectively), but expression of MCL1 was upregulated in creta sites (p = 0.039). MCL1 expression was significantly downregulated in HTR-8/SVneo cells overexpressing miR-29a/b/c (p = 0.002, 0.008, and 0.013, respectively). Luciferase activity assays revealed that miR-29a/b/c directly target the 3′ untranslated region of MCL1 in 293T cells. Over-expression of miR-29a/b/c induced apoptosis in the HTR-8/SVneo trophoblast cell line. Moreover, histopathological evaluation revealed that the number of implantation site intermediate trophoblast (ISIT) cells was increased in creta sites and that these cells were positive for MCL1. ConclusionsOur results demonstrate that in placenta accreta, miR-29a/b/c inhibits apoptosis of ISIT cells by targeting MCL1. These findings provide new insights into the pathogenesis of placenta accreta.