Augmenting NK cell-based immunotherapy by targeting mitochondrial apoptosis.

Interest in harnessing natural killer (NK) cells for cancer immunotherapy is rapidly growing. However, efficacy of NK cell-based immunotherapy remains limited in most trials. Strategies to augment the killing efficacy of NK cells are thus much needed. In the current study, we found that mitochondrial apoptosis (mtApoptosis) pathway is essential for efficient NK killing, especially at physiologically relevant effector-to-target ratios. Furthermore, NK cells can prime cancer cells for mtApoptosis and mitochondrial priming status affects cancer-cell susceptibility to NK-mediated killing. Interestingly, pre-activating NK cells confers on them resistance to BH3 mimetics. Combining BH3 mimetics with NK cells synergistically kills cancer cells in vitro and suppresses tumor growth in vivo. The ideal BH3 mimetic to use in such an approach can be predicted by BH3 profiling. We herein report a rational and precision strategy to augment NK-based immunotherapy, which may be adaptable to T cell-based immunotherapies as well.

Small Molecules Co-targeting CKIα and the Transcriptional Kinases CDK7/9 Control AML in Preclinical Models.

CKIα ablation induces p53 activation, and CKIα degradation underlies the therapeutic effect of lenalidomide in a pre-leukemia syndrome. Here we describe the development of CKIα inhibitors, which co-target the transcriptional kinases CDK7 and CDK9, thereby augmenting CKIα-induced p53 activation and its anti-leukemic activity. Oncogene-driving super-enhancers (SEs) are highly sensitive to CDK7/9 inhibition. We identified multiple newly gained SEs in primary mouse acute myeloid leukemia (AML) cells and demonstrate that the inhibitors abolish many SEs and preferentially suppress the transcription elongation of SE-driven oncogenes. We show that blocking CKIα together with CDK7 and/or CDK9 synergistically stabilize p53, deprive leukemia cells of survival and proliferation-maintaining SE-driven oncogenes, and induce apoptosis. Leukemia progenitors are selectively eliminated by the inhibitors, explaining their therapeutic efficacy with preserved hematopoiesis and leukemia cure potential; they eradicate leukemia in MLL-AF9 and Tet2-/-;Flt3ITD AML mouse models and in several patient-derived AML xenograft models, supporting their potential efficacy in curing human leukemia.

Intrinsically determined turnover underlies broad heterogeneity in plasma-cell lifespan.

Antibodies produced by antibody-secreting plasma cells (ASCs) underlie multiple forms of long-lasting immunity. Here we examined the mechanisms regulating ASC turnover and persistence using a genetic reporter to time-stamp ASCs. This approach revealed ASC lifespans as heterogeneous and falling on a continuum, with only a small fraction surviving for >60 days. ASC longevity past 60 days was independent of isotype but correlated with a phenotype that developed progressively and ultimately associated with an underlying "long-lived" ASC (LL ASC)-enriched transcriptional program. While some of the differences between LL ASCs and other ASCs appeared to be acquired with age, other features were shared with some younger ASCs, such as high CD138 and CD93. Turnover was unaffected by altered ASC production, arguing against competition for niches as a major driver of turnover. Thus, ASC turnover is set by intrinsic lifespan limits, with steady-state population dynamics governed by niche vacancy rather than displacement.

Anti-apoptotic MCL-1 promotes long-chain fatty acid oxidation through interaction with ACSL1.

MCL-1 is essential for promoting the survival of many normal cell lineages and confers survival and chemoresistance in cancer. Beyond apoptosis regulation, MCL-1 has been linked to modulating mitochondrial metabolism, but the mechanism(s) by which it does so are unclear. Here, we show in tissues and cells that MCL-1 supports essential steps in long-chain (but not short-chain) fatty acid ß-oxidation (FAO) through its binding to specific long-chain acyl-coenzyme A (CoA) synthetases of the ACSL family. ACSL1 binds to the BH3-binding hydrophobic groove of MCL-1 through a non-conventional BH3-domain. Perturbation of this interaction, via genetic loss of Mcl1, mutagenesis, or use of selective BH3-mimetic MCL-1 inhibitors, represses long-chain FAO in cells and in mouse livers and hearts. Our findings reveal how anti-apoptotic MCL-1 facilitates mitochondrial metabolism and indicate that disruption of this function may be associated with unanticipated cardiac toxicities of MCL-1 inhibitors in clinical trials.

Dynamic Regulation of Long-Chain Fatty Acid Oxidation by a Noncanonical Interaction between the MCL-1 BH3 Helix and VLCAD.

MCL-1 is a BCL-2 family protein implicated in the development and chemoresistance of human cancer. Unlike its anti-apoptotic homologs, Mcl-1 deletion has profound physiologic consequences, indicative of a broader role in homeostasis. We report that the BCL-2 homology 3 (BH3) α helix of MCL-1 can directly engage very long-chain acyl-CoA dehydrogenase (VLCAD), a key enzyme of the mitochondrial fatty acid ß-oxidation (FAO) pathway. Proteomic analysis confirmed that the mitochondrial matrix isoform of MCL-1 (MCL-1Matrix) interacts with VLCAD. Mcl-1 deletion, or eliminating MCL-1Matrix alone, selectively deregulated long-chain FAO, causing increased flux through the pathway in response to nutrient deprivation. Transient elevation in MCL-1 upon serum withdrawal, a striking increase in MCL-1 BH3/VLCAD interaction upon palmitic acid titration, and direct modulation of enzymatic activity by the MCL-1 BH3 α helix are consistent with dynamic regulation. Thus, the MCL-1 BH3 interaction with VLCAD revealed a separable, gain-of-function role for MCL-1 in the regulation of lipid metabolism.

Novel markers of MCL1 inhibitor sensitivity in triple-negative breast cancer cells.

Triple-negative breast cancer (TNBC) is an aggressive breast cancer sub-type with limited treatment options and poor prognosis. Currently, standard treatments for TNBC include surgery, chemotherapy, and anti-PDL1 therapy. These therapies have limited efficacy in advanced stages. Myeloid-cell leukemia 1 (MCL1) is an anti-apoptotic BCL2 family protein. High expression of MCL1 contributes to chemotherapy resistance and is associated with a worse prognosis in TNBC. MCL1 inhibitors are in clinical trials for TNBC, but response rates to these inhibitors can vary and predictive markers are lacking. Currently, we identified a 4-member (AXL, ETS1, IL6, EFEMP1) gene signature (GS) that predicts MCL1 inhibitor sensitivity in TNBC cells. Factors encoded by these genes regulate signaling pathways to promote MCL1 inhibitor resistance. Small molecule inhibitors of the GS factors can overcome resistance and sensitize otherwise resistant TNBC cells to MCL1 inhibitor treatment. These findings offer insights into potential therapeutic strategies and tumor stratification for MCL1 inhibitor use in TNBC.

The HTLV-I oncoprotein Tax inactivates the tumor suppressor FBXW7.

Human T-cell leukemia virus type 1 (HTLV-I) is the etiological agent of adult T-cell leukemia (ATL). Mutational analysis has demonstrated that the tumor suppressor, F-box and WD repeat domain containing 7 (FBXW7/FBW7/CDC4), is mutated in primary ATL patients. However, even in the absence of genetic mutations, FBXW7 substrates are stabilized in ATL cells, suggesting additional mechanisms can prevent FBXW7 functions. Here, we report that the viral oncoprotein Tax represses FBXW7 activity, resulting in the stabilization of activated Notch intracellular domain, c-MYC, Cyclin E, and myeloid cell leukemia sequence 1 (BCL2-related) (Mcl-1). Mechanistically, we demonstrate that Tax directly binds to FBXW7 in the nucleus, effectively outcompeting other targets for binding to FBXW7, resulting in decreased ubiquitination and degradation of FBXW7 substrates. In support of the nuclear role of Tax, a non-degradable form of the nuclear factor kappa B subunit 2 (NFκB2/p100) was found to delocalize Tax to the cytoplasm, thereby preventing Tax interactions with FBXW7 and Tax-mediated inhibition of FBXW7. Finally, we characterize a Tax mutant that is unable to interact with FBXW7, unable to block FBXW7 tumor suppressor functions, and unable to effectively transform fibroblasts. These results demonstrate that HTLV-I Tax can inhibit FBXW7 functions without genetic mutations to promote an oncogenic state. These results suggest that Tax-mediated inhibition of FBXW7 is likely critical during the early stages of the cellular transformation process. IMPORTANCE: F-box and WD repeat domain containing 7 (FBXW7), a critical tumor suppressor of human cancers, is frequently mutated or epigenetically suppressed. Loss of FBXW7 functions is associated with stabilization and increased expression of oncogenic factors such as Cyclin E, c-Myc, Mcl-1, mTOR, Jun, and Notch. In this study, we demonstrate that the human retrovirus human T-cell leukemia virus type 1 oncoprotein Tax directly interacts with FBXW7, effectively outcompeting other targets for binding to FBXW7, resulting in decreased ubiquitination and degradation of FBXW7 cellular substrates. We further demonstrate that a Tax mutant unable to interact with and inactivate FBXW7 loses its ability to transform primary fibroblasts. Collectively, our results describe a novel mechanism used by a human tumor virus to promote cellular transformation.

TCEB3 initiates ovarian cancer apoptosis by mediating ubiquitination and degradation of MCL-1.

Platinum resistance remains a major contributor to the poor prognosis of ovarian cancer. Anti-apoptotic protein myeloid cell leukemia-1 (MCL-1) has emerged as a promising target for overcoming drug resistance, but different cancer cells utilize distinct protein degradation pathways to alter MCL-1 level. We systematically investigated E3 ligases to identify novel candidates that mediate platinum resistance in ovarian cancer. Transcription Elongation Factor B (TCEB3) has been identified as a novel E3 ligase recognition subunit that targets MCL-1 in the cytoplasm during platinum treatment other than its traditional function of targeting the Pol II in the nuclear compartment. TCEB3 expression is downregulated in platinum-resistant cell lines and this low expression is associated with poor prognosis. The ubiquitination of MCL-1 induced by TCEB3 leads to cell death in ovarian cancer. Moreover, platinum treatment increased the cytoplasm proportion of TCEB3, and the cytoplasm localization of TCEB3 is important for its targeting of MCL-1. This study emphasizes the dual function of TCEB3 in homeostasis maintenance and in cell fate determination under different conditions, and provides a new insight into drug resistance in ovarian cancer.

Clitocine enhances the drug sensitivity of colon cancer cells by promoting FBXW7-mediated MCL-1 degradation via inhibiting the A2B/cAMP/ERK axis.

Chemotherapy resistance to colon cancer is an unavoidable obstacle in the clinical management of the disease. Clitocine, an adenosine analog, played a significant role in the chemosensitivity of human colon cancer cells by promoting MCL-1 protein degradation. However, the detailed mechanism remains to be further elucidated. We found that clitocine up-regulates the expression of FBXW7, a ubiquitin ligase involved in the MCL-1 degradation. Transcriptome sequencing analysis revealed that clitocine significantly inhibits the cAMP and ERK downstream signaling pathways in colon cancer cells, thereby enhancing FBXW7 expression and subsequently promoting the ubiquitination degradation of MCL-1 protein. We verified that clitocine regulated intracellular cAMP levels by competitive binding with the adenosine receptor A2B. Molecular docking assay also verified the binding relationship. By decreasing intracellular cAMP levels, clitocine blocks the activation of downstream signaling pathways, which ultimately enhances the drug sensitivity of colon cancer cells through increased FBXW7 expression due to the inhibition of its promoter DNA methylation. Both knock-out of adenosine receptor A2B and Br-cAMP treatment can effectively attenuate the function of clitocine in vitro and in vivo. This study clarified that clitocine enhanced the drug sensitivity of colon cancer cells by promoting FBXW7-mediated MCL-1 degradation via inhibiting the A2B/cAMP/ERK axis, providing further knowledge of the clinical application for clitocine.

Structural transitions in TCTP tumor protein upon binding to the anti-apoptotic protein family member Mcl-1.

Translationally Controlled Tumor Protein (TCTP) serves as a pro-survival factor in tumor cells, inhibiting the mitochondrial apoptosis pathway by enhancing the function of anti-apoptotic Bcl-2 family members Mcl-1 and Bcl-xL. TCTP specifically binds to Bcl-xL, preventing Bax-dependent Bcl-xL-induced cytochrome c release, and it reduces Mcl-1 turnover by inhibiting its ubiquitination, thereby decreasing Mcl-1-mediated apoptosis. TCTP harbors a BH3-like motif that forms a ß-strand buried in the globular domain of the protein. In contrast, the crystal structure of the TCTP BH3-like peptide in complex with the Bcl-2 family member Bcl-xL reveals an α-helical conformation for the BH3-like motif, suggesting significant structural changes upon complex formation. Employing biochemical and biophysical methods, including limited proteolysis, circular dichroism, NMR, and SAXS, we describe the TCTP complex with the Bcl-2 homolog Mcl-1. Our findings demonstrate that full-length TCTP binds to the BH3 binding groove of Mcl-1 via its BH3-like motif, experiencing conformational exchange at the interface on a micro- to milli-second timescale. Concurrently, the TCTP globular domain becomes destabilized, transitioning into a molten-globule state. Furthermore, we establish that the non-canonical residue D16 within the TCTP BH3-like motif reduces stability while enhancing the dynamics of the intermolecular interface. In conclusion, we detail the structural plasticity of TCTP and discuss its implications for partner interactions and future anticancer drug design strategies aimed at targeting TCTP complexes.

FLT3 and IRAK4 Inhibitor Emavusertib in Combination with BH3-Mimetics in the Treatment of Acute Myeloid Leukemia.

Targeting the FLT3 receptor and the IL-1R associated kinase 4 as well as the anti-apoptotic proteins MCL1 and BCL2 may be a promising novel approach in the treatment of acute myeloid leukemia (AML). The FLT3 and IRAK4 inhibitor emavusertib (CA4948), the MCL1 inhibitor S63845, the BCL2 inhibitor venetoclax, and the HSP90 inhibitor PU-H71 were assessed as single agents and in combination for their ability to induce apoptosis and cell death in leukemic cells in vitro. AML cells represented all major morphologic and molecular subtypes, including FLT3-ITD and NPM1 mutant AML cell lines and a variety of patient-derived AML cells. Emavusertib in combination with MCL1 inhibitor S63845 or BCL2 inhibitor venetoclax induced cell cycle arrest and apoptosis in MOLM-13 cells. In primary AML cells, the response to emavusertib was associated with the presence of the FLT3 gene mutation with an allelic ratio >0.5 and the presence of NPM1 gene mutations. S63845 was effective in all tested AML cell lines and primary AML samples. Blast cell percentage was positively associated with the response to CA4948, S63845, and venetoclax, with elevated susceptibility of primary AML with blast cell fraction >80%. Biomarkers of the response to venetoclax included the blast cell percentage and bone marrow infiltration rate, as well as the expression levels of CD11b, CD64, and CD117. Elevated susceptibility to CA4948 combination treatments with S63845 or PU-H71 was associated with FLT3-mutated AML and CD34 < 30%. The combination of CA4948 and BH3-mimetics may be effective in the treatment in FLT3-mutated AML with differential target specificity for MCL1 and BCL2 inhibitors. Moreover, the combination of CA4948 and PU-H71 may be a candidate combination treatment in FLT3-mutated AML.

Initial AXL and MCL-1 inhibition contributes to abolishing lazertinib tolerance in EGFR-mutant lung cancer cells.

Lazertinib, a novel third-generation epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI), demonstrates marked efficacy in EGFR-mutant lung cancer. However, resistance commonly develops, prompting consideration of therapeutic strategies to overcome initial drug resistance mechanisms. This study aimed to elucidate the adaptive resistance to lazertinib and advocate novel combination treatments that demonstrate efficacy in preventing resistance as a first-line treatment for EGFR mutation-positive NSCLC. We found that AXL knockdown significantly inhibited lung cancer cell viability in the presence of lazertinib, indicating that AXL activation contributes to lazertinib resistance. However, long-term culture with a combination of lazertinib and AXL inhibitors led to residual cell proliferation and increased the MCL-1 expression level, which was mediated by the nuclear translocation of the transcription factor YAP. Triple therapy with an MCL-1 or YAP inhibitor in combination with lazertinib and an AXL inhibitor significantly reduced cell viability and increased the apoptosis rate. These results demonstrate that AXL and YAP/MCL-1 signals contribute to adaptive lazertinib resistance in EGFR-mutant lung cancer cells, suggesting that the initial dual inhibition of AXL and YAP/MCL-1 might be a highly effective strategy in eliminating lazertinib-resistant cells.

Synergistic cytotoxicity of decitabine and YM155 in leukemia cells through upregulation of SLC35F2 and suppression of MCL1 and survivin expression.

The hypomethylation agent decitabine (DAC), in combination with other apoptosis inducers, is considered a potential modality for cancer treatment. We investigated the mechanism underlying the combined cytotoxicity of DAC and YM155 in acute myeloid leukemia (AML) cells because of increasing evidence that YM155 induces apoptosis in cancer cells. Co-administration of DAC and YM155 resulted in synergistic cytotoxicity in AML U937 cells, which was characterized by the induction of apoptosis, NOXA-dependent degradation of MCL1 and survivin, and depolarization of mitochondria. Restoration of MCL1 or survivin expression attenuated DAC/YM155-induced U937 cell death. DAC initiated AKT and p38 MAPK phosphorylation in a Ca2+/ROS-dependent manner, thereby promoting autophagy-mediated degradation of ß-TrCP mRNA, leading to increased Sp1 expression. DAC-induced Sp1 expression associated with Ten-eleven-translocation (TET) dioxygenases and p300 was used to upregulate the expression of SLC35F2. Simultaneously, the activation of p38 MAPK induced by DAC, promoted CREB-mediated NOXA expression, resulting in survivin and MCL1 degradation. The synergistic cytotoxicity of DAC and YM155 in U937 cells was dependent on elevated SLC35F2 expression. Additionally, YM155 facilitated DAC-induced degradation of MCL1 and survivin. A similar mechanism explained DAC/YM155-mediated cytotoxicity in AML HL-60 cells. Our data demonstrated that the synergistic cytotoxicity of DAC and YM155 in AML cell lines U937 and HL-60 is dependent on AKT- and p38 MAPK-mediated upregulation of SLC35F2 and p38 MAPK-mediated degradation of survivin and MCL1. This indicates that a treatment regimen that amalgamates YM155 and DAC may be beneficial for AML.

Regulatory axis of circular RNA DTNB, microRNA-485-5p, and myeloid cell leukemia 1 attenuates inflammation and apoptosis in caerulein-treated AR42J cells.

Acute pancreatitis (AP) is an inflammatory disease of the pancreas and the main cause of hospital admissions for gastrointestinal diseases. Here, the work studied the circular RNA DTNB/microRNA-485-5p/MCL1 axis in AP and hoped to unravel the related mechanism. Caerulein exposure replicated an AP model in AR42J cells, and caerulein-mediated expression of circDTNB, miR-485-5p, and MCL1 was recorded. After exposure, cells were intervened with transfection plasmids and tested for LDH release, apoptosis, and inflammation. To determine the interwork of circDTNB, miR-485-5p, and MCL1, prediction results and verification experiments were conducted. Caerulein exposure reduced circDTNB and MCL1, while elevated miR-485-5p levels in AR42J cells. Upregulating circDTNB protected AR42J cells from caerulein-induced LDH cytotoxicity, apoptosis, and inflammation, but circDTNB upregulation-induced protections could be muffled by inhibiting MCL1. On the contrary, downregulating circDTNB further damaged AR42J cells under caerulein exposure, however, this phenomenon could be partially rescued after silencing miR-485-5p. miR-485-5p was mechanistically verified to be a target of circDTNB to mediate MCL1. Overall, the circDTNB/miR-485-5p/MCL1 axis protects inflammatory response and apoptosis in caerulein-exposed AR42J cells, promisingly identifying circDTNB as a novel molecule for AP treatment.

FBXW7 regulates the sensitivity of imatinib in gastrointestinal stromal tumors by targeting MCL1.

BACKGROUND: Imatinib contributes to improving prognosis of high-risk or unresectable gastrointestinal stromal tumors (GISTs). As therapeutic efficacy is limited by imatinib resistance and toxicity, the exploration of predictive markers of imatinib therapeutic efficacy that enables patients to utilize more effective therapeutic strategies remains urgent. METHODS: The correlation between FBXW7 and imatinib resistance via FBXW7-MCL1 axis was evaluated in vitro and in vivo experiments. The significance of FBXW7 as a predictor of imatinib treatment efficacy was examined in 140 high-risk patients with GISTs. RESULTS: The ability of FBXW7 to predict therapeutic efficacy of adjuvant imatinib in high-risk GIST patients was determined through 5-year recurrence-free survival (RFS) rates analysis and multivariate analysis. FBXW7 affects imatinib sensitivity by regulating apoptosis in GIST-T1 cells. FBXW7 targets MCL1 to regulate apoptosis. MCL1 involves in the regulation of imatinib sensitivity through inhibiting apoptosis in GIST-T1 cells. FBXW7 regulates imatinib sensitivity by down-regulating MCL1 to enhance imatinib-induced apoptosis in vitro. FBXW7 regulates imatinib sensitivity of GIST cells by targeting MCL1 to predict efficacy of imatinib treatment in vivo. CONCLUSIONS: FBXW7 regulates imatinib sensitivity by inhibiting MCL1 to enhance imatinib-induced apoptosis in GIST, and predicts efficacy of imatinib treatment in high-risk GIST patients treated with imatinib.

Discovery and development of thiazolidine-2,4-dione derivatives as Bcl-2/Mcl-1 dual inhibitors.

Increasing the levels of antiapoptotic Bcl-2 proteins is an important way that cancer cells utilize to get out of apoptosis, underscoring their significance as promising targets for anticancer therapies. Lately, a primary compound 1 bearing thiazolidine-2,4-dione was discovered to exhibit comparable Mcl-1 inhibitory activity in comparison to WL-276. Herein, thirty-nine thiazolidine-2,4-dione analogs were yielded through incorporating different biphenyl moieties (R1), amino acid side chains (R2) and sulfonamides (R3) on 1. The findings indicated that certain compounds exhibited favorable inhibitory effects against Bcl-2/Mcl-1, while demonstrating limited or negligible binding affinity towards Bcl-xL. In particular, compounds 16 and 20 exhibited greater Bcl-2/Mcl-1 inhibition compared to AT-101, WL-276 and 1. Moreover, they demonstrated notable antiproliferative effects and significantly induced apoptosis in U937 cells. The western blot and co-immunoprecipitation assays confirmed that 20 could induce alterations in the expression of apoptosis-associated proteins to result in apoptosis through on-target Bcl-2 and Mcl-1 inhibition. In addition, 20 exhibited favorable stability profiles in both rat plasma and rat liver microsomes. In total, 20 could be used as a promising compound to discover Bcl-2/Mcl-1 dual inhibitors with favorable therapeutic properties.

Targeting cytohesin-1 suppresses acute myeloid leukemia progression and overcomes resistance to ABT-199.

Adhesion molecules play essential roles in the homeostatic regulation and malignant transformation of hematopoietic cells. The dysregulated expression of adhesion molecules in leukemic cells accelerates disease progression and the development of drug resistance. Thus, targeting adhesion molecules represents an attractive anti-leukemic therapeutic strategy. In this study, we investigated the prognostic role and functional significance of cytohesin-1 (CYTH1) in acute myeloid leukemia (AML). Analysis of AML patient data from the GEPIA and BloodSpot databases revealed that CYTH1 was significantly overexpressed in AML and independently correlated with prognosis. Functional assays using AML cell lines and an AML xenograft mouse model confirmed that CYTH1 depletion significantly inhibited the adhesion, migration, homing, and engraftment of leukemic cells, delaying disease progression and prolonging animal survival. The CYTH1 inhibitor SecinH3 exerted in vitro and in vivo anti-leukemic effects by disrupting leukemic adhesion and survival programs. In line with the CYTH1 knockdown results, targeting CYTH1 by SecinH3 suppressed integrin-associated adhesion signaling by reducing ITGB2 expression. SecinH3 treatment efficiently induced the apoptosis and inhibited the growth of a panel of AML cell lines (MOLM-13, MV4-11 and THP-1) with mixed-lineage leukemia gene rearrangement, partly by reducing the expression of the anti-apoptotic protein MCL1. Moreover, we showed that SecinH3 synergized with the BCL2-selective inhibitor ABT-199 (venetoclax) to inhibit the proliferation and promote the apoptosis of ABT-199-resistant leukemic cells. Taken together, our results not only shed light on the role of CYTH1 in cell-adhesion-mediated leukemogenesis but also propose a novel combination treatment strategy for AML.

TRAF6 Restricts p53 Mitochondrial Translocation, Apoptosis, and Tumor Suppression.

Mitochondrial p53 is involved in apoptosis and tumor suppression. However, its regulation is not well studied. Here, we show that TRAF6 E3 ligase is a crucial factor to restrict mitochondrial translocation of p53 and spontaneous apoptosis by promoting K63-linked ubiquitination of p53 at K24 in cytosol, and such ubiquitination limits the interaction between p53 and MCL-1/BAK. Genotoxic stress reduces this ubiquitination in cytosol by S13/T330 phosphorylation-dependent translocation of TRAF6 from cytosol to nucleus, where TRAF6 also facilitates the K63-linked ubiquitination of nuclear p53 and its transactivation by recruiting p300 for p53 acetylation. Functionally, K63-linked ubiquitination of p53 compromised p53-mediated apoptosis and tumor suppression. Colorectal cancer samples with WT p53 reveal that TRAF6 overexpression negatively correlates with apoptosis and predicts poor response to chemotherapy and radiotherapy. Together, our study identifies TRAF6 as a critical gatekeeper to restrict p53 mitochondrial translocation, and such mechanism may contribute to tumor development and drug resistance.

The Role of miR-29a and miR-143 on the Anti-apoptotic MCL-1/cIAP-2 Genes Expression in EGFR Mutated Non-small Cell Lung Carcinoma Patients.

The survival rate of lung cancer is low due to the high frequency of drug resistance in patients with mutations in the driver genes. Overexpression of anti-apoptotic genes is one of the most prominent features of tumor drug resistance. EGFR signaling induces the expression of anti-apoptotic genes. Also, microRNAs (miRNAs) have a critical role in regulating biological functions such as apoptosis; a process mostly eluded in cancer progression. The mutation screening was performed on one thousand non-small cell lung carcinoma patients to enroll clinical samples in this study. Bioinformatics analysis predicted that miRNAs (miR-29a, miR-143) might regulate MCL-1 and cIAP-2 expression. We investigated the expression of MCL-1, cIAP-2, miR-29a, and miR-143 encoding genes in adenocarcinoma patients with or without EGFR mutations before treatment. The potential role of miR-29a and miR-143 on gene expression was evaluated by overexpression and luciferase assays in HEK-293T cells. EGFR mutations were found in 262 patients (26.2%) with a greater incidence in females (36.23% vs. 20.37%, P = 0.001). The expression levels of MCL-1 and cIAP-2 genes in patients with mutated EGFR were higher than those of wild-type EGFR. In contrast, compared to those of patients with wild-type EGFR, the expression levels of miR-29a and miR-143 were lower in the patients carrying EGFR mutations. In cell culture, overexpression of miR-29a and miR-143 significantly downregulated the expression of MCL-1 and cIAP-2. Dual-luciferase reporter experiments confirmed that miR-29a and miR-143 target MCL-1 and cIAP-2 mRNAs, respectively. Our results suggest that upregulation of EGFR signaling in lung cancer cells may increase anti-apoptotic MCL-1 and cIAP-2 gene expression, possibly through downregulation of miR-29a-3p and miR-143-3p.

The Role of microRNAs Related to Apoptosis for N-Methyl-d-Aspartic Acid-Induced Neuronal Cell Death in the Murine Retina.

Glaucoma is one of the leading causes of acquired blindness and characterized by retinal ganglion cell (RGC) death. MicroRNAs are small noncoding RNAs that degrade their target mRNAs. Apoptosis is one of the common mechanisms leading to neuronal death in many neurodegenerative diseases, including glaucoma. In the present study, we identified microRNAs that modulate RGC death caused by the intravitreal injection of N-methyl-d-aspartic acid (NMDA). We found an upregulation of miR-29b and downregulation of miR-124 in the retina of the NMDA-injected eyes. The intravitreal injection of an miR-29b inhibitor 18 h before NMDA injection reduced RGC death and the downregulation of myeloid cell leukemia 1 (MCL-1), an anti-apoptotic factor, induced by intravitreal NMDA. The intravitreal injection of an miR-124 mimic 18 h before NMDA injection also reduced RGC death and the upregulation of B-cell/chronic lymphocytic leukemia lymphoma 2 (bcl-2)-associated X protein (Bax) and bcl-2 interacting protein (Bim), pro-apoptotic factors, induced by intravitreal NMDA. These data suggest that expressional changes in microRNA are involved in the excitotoxicity of RGCs, and that complement and/or inhibition of microRNA may be a potential therapeutic approach for the diseases related to the excitotoxicity of RGCs, such as glaucoma and retinal central artery occlusion.