Calcium-binding protein 7 expressed in muscle negatively regulates age-related degeneration of neuromuscular junctions in mice.

The neuromuscular junction (NMJ) forms centrally in myotubes and, as the only synapse between motor neuron and myotube, are indispensable for motor activity. The midmuscle formation of NMJs, including midmuscle-restricted expression of NMJ-related genes, is governed by the muscle-specific kinase (MuSK). However, mechanisms underlying MuSK-mediated signaling are unclear. Here, we find that the Calcium-binding protein 7 (Cabp7) gene shows midmuscle-restricted expression, and muscle-specific depletion of Cabp7 in mice accelerated age-related NMJ degeneration, muscle weakness/atrophy, and motor dysfunction. Surprisingly, forced expression in muscle of CIP, an inhibitory peptide of the negative regulator of NMJ formation cyclin-dependent kinase 5 (Cdk5), restored NMJ integrity and muscle strength, and healed muscle atrophy in muscle-specific Cabp7-deficient mice, which showed increased muscle expression of the Cdk5 activator p25. These findings together demonstrate that MuSK-mediated signaling induces muscle expression of Cabp7, which suppresses age-related NMJ degeneration likely by attenuating p25 expression, providing insights into prophylactic/therapeutic intervention against age-related motor dysfunction.

[Molecular Mechanisms Underlying the Formation and Maintenance of Neuromuscular Junctions and Development of NMJ-targeted Therapeutics].

Skeletal muscle is an essential organ for the motor functions and its defects are associated with functional impairments of other organs including brain. Muscle contraction is the fundamental skeletal muscle function and is strictly controlled by motor neuron, which requires neuromuscular junction (NMJ), a chemical synapse between motor nerve terminal and myotube (myofiber). Defects in NMJ cause various functional abnormalities of skeletal muscle including muscle weakness. This review presents an overview of the current understanding of signaling in NMJ formation and maintenance in skeletal muscle and the development of NMJ-targeted therapeutics.

LONRF2 is a protein quality control ubiquitin ligase whose deficiency causes late-onset neurological deficits.

Protein misfolding is a major factor of neurodegenerative diseases. Post-mitotic neurons are highly susceptible to protein aggregates that are not diluted by mitosis. Therefore, post-mitotic cells may have a specific protein quality control system. Here, we show that LONRF2 is a bona fide protein quality control ubiquitin ligase induced in post-mitotic senescent cells. Under unperturbed conditions, LONRF2 is predominantly expressed in neurons. LONRF2 binds and ubiquitylates abnormally structured TDP-43 and hnRNP M1 and artificially misfolded proteins. Lonrf2-/- mice exhibit age-dependent TDP-43-mediated motor neuron (MN) degeneration and cerebellar ataxia. Mouse induced pluripotent stem cell-derived MNs lacking LONRF2 showed reduced survival, shortening of neurites and accumulation of pTDP-43 and G3BP1 after long-term culture. The shortening of neurites in MNs from patients with amyotrophic lateral sclerosis is rescued by ectopic expression of LONRF2. Our findings reveal that LONRF2 is a protein quality control ligase whose loss may contribute to MN degeneration and motor deficits.

SETD2 regulates chromatin accessibility and transcription to suppress lung tumorigenesis.

SETD2, a H3K36 trimethyltransferase, is the most frequently mutated epigenetic modifier in lung adenocarcinoma, with a mutation frequency of approximately 9%. However, how SETD2 loss of function promotes tumorigenesis remains unclear. Using conditional Setd2-KO mice, we demonstrated that Setd2 deficiency accelerated the initiation of KrasG12D-driven lung tumorigenesis, increased tumor burden, and significantly reduced mouse survival. An integrated chromatin accessibility and transcriptome analysis revealed a potentially novel tumor suppressor model of SETD2 in which SETD2 loss activates intronic enhancers to drive oncogenic transcriptional output, including the KRAS transcriptional signature and PRC2-repressed targets, through regulation of chromatin accessibility and histone chaperone recruitment. Importantly, SETD2 loss sensitized KRAS-mutant lung cancer to inhibition of histone chaperones, the FACT complex, or transcriptional elongation both in vitro and in vivo. Overall, our studies not only provide insight into how SETD2 loss shapes the epigenetic and transcriptional landscape to promote tumorigenesis, but they also identify potential therapeutic strategies for SETD2 mutant cancers.

Loss of Dok-3 in Non-tumor Cells Induces Malignant Transformation of Benign Epithelial Tumor Cells of the Intestine.

The fundamental difference between benign and malignant tumors lies in their invasive ability. It is believed that malignant conversion of benign tumor cells is induced by a tumor cell-intrinsic accumulation of driver gene mutations. Here, we found that disruption of the Dok-3 tumor suppressor gene led to malignant progression in the intestinal benign tumor model ApcMin/+ mice. However, Dok-3 gene expression was undetectable in epithelial tumor cells and the transplantation of bone marrow cells lacking the Dok-3 gene-induced malignant conversion of epithelial tumor cells in ApcMin/+ mice, indicating a previously unrecognized tumor cell-extrinsic mechanism. Moreover, the Dok-3 loss-induced tumor invasion in ApcMin/+ mice required CD4+ and CD8+ T lymphocytes, but not B lymphocytes. Finally, whole-genome sequencing showed an indistinguishable pattern and level of somatic mutations in tumors irrespective of the Dok-3 gene mutation in ApcMin/+ mice. Together, these data indicate that Dok-3 deficiency is a tumor-extrinsic driving force of malignant progression in ApcMin/+ mice, providing a novel insight into microenvironments in tumor invasion. Significance: This study uncovers tumor cell-extrinsic cues that can induce malignant conversion of benign tumors without intensifying mutagenesis in tumors, a novel concept potentially providing a new therapeutic target in malignancy.

EMT-inducing transcription factor ZEB1-associated resistance to the BCL-2/BCL-XL inhibitor is overcome by BIM upregulation in ovarian clear cell carcinoma cells.

Ovarian clear cell carcinoma (OCCC) is an aggressive subtype of epithelial ovarian cancer, which generally exhibits chemoresistance. Effective therapy for OCCC is currently unavailable, requiring the development of new therapeutic strategies. ABT-263 (navitoclax), an inhibitor of the anti-apoptotic BCL-2/BCL-XL, has a potent ability of inducing death in cancer cells; however, the therapeutic effect of ABT-263 in OCCC remains unclear. Epithelial cells undergo epithelial-mesenchymal transition (EMT) to acquire a mesenchymal phenotype, which is known to contribute to the development of resistance against therapeutic agents. In this study, we revealed that the sensitivity of OCCC cells to ABT-263 was associated with the epithelial/mesenchymal status of the cells. While the OCCC cells with an epithelial phenotype were ABT-263-sensitive, those with a mesenchymal phenotype were ABT-263-resistant, which was accompanied by an insufficient expression of the pro-apoptotic BH3 protein BIM. Mechanistically, the EMT-inducing transcription factor, ZEB1 down-regulated BIM transcription by binding to BIM promoter, resulting in resistance to ABT-263. It is noteworthy that ZEB1-associated ABT-263 resistance was overcome by an HDAC inhibitor, FK228 (romidepsin), through the up-regulation of BIM. In summary, our study provides evidence for a mechanism for ABT-263 resistance in OCCC cells as well as a potential therapeutic strategy to overcome it.

Loss of C/EBPδ enhances apoptosis of intestinal epithelial cells and exacerbates experimental colitis in mice.

Inflammatory bowel diseases (IBDs) are characterized by chronic inflammation involving intestinal tissue damage, which include ulcerative colitis and Crohn's disease as major entities. Accumulating evidence suggests that excessive apoptosis of intestinal epithelial cells (IECs) contributes to the development of IBD. It was recently reported that the transcription factor CCAAT/enhancer-binding protein delta (C/EBPδ) is involved in inflammation; however, its role in colitis remains unclear. Here, we found that C/EBPδ knockout mice showed enhanced susceptibility to dextran sodium sulfate (DSS)-induced colitis, a mouse model of IBD, which was associated with severe colonic inflammation and mucosal damage with increased IEC apoptosis. Additionally, DSS stimulation induced increased expression of pro-apoptotic BH3-only protein Bim in the colon of C/EBPδ knockout mice. Collectively, our findings demonstrate that C/EBPδ plays an essential role in suppressing DSS-induced colitis, likely by attenuating IEC apoptosis.

BH3-Dependent and Independent Activation of BAX and BAK in Mitochondrial Apoptosis.

Mitochondria play key roles in mammalian apoptosis, a highly regulated genetic program of cell suicide. Multiple apoptotic signals culminate in mitochondrial outer membrane permeabilization (MOMP), which not only couples the mitochondria to the activation of caspases but also initiates caspase-independent mitochondrial dysfunction. The BCL-2 family proteins are central regulators of MOMP. Multidomain pro-apoptotic BAX and BAK are essential effectors responsible for MOMP, whereas anti-apoptotic BCL-2, BCL-XL, and MCL-1 preserve mitochondrial integrity. The third BCL-2 subfamily of proteins, BH3-only molecules, promotes apoptosis by either activating BAX and BAK or inactivating BCL-2, BCL-XL, and MCL-1. Through an interconnected hierarchical network of interactions, the BCL-2 family proteins integrate developmental and environmental cues to dictate the survival versus death decision of cells by regulating the integrity of the mitochondrial outer membrane. Over the past 30 years, research on the BCL-2-regulated apoptotic pathway has not only revealed its importance in both normal physiological and disease processes, but has also resulted in the first anti-cancer drug targeting protein-protein interactions.

Eicosapentaenoic acid attenuates obesity-related hepatocellular carcinogenesis.

Non-alcoholic fatty liver disease (NAFLD), the hepatic manifestation of obesity, is an emerging risk factor for hepatocellular carcinoma (HCC). Accumulating evidence has shown that chronic inflammation represents a plausible link between obesity and HCC and that the pro-inflammatory cytokine interleukin (IL)-6 contributes to the development of obesity-related HCC. In the present study, we aimed to examine the therapeutic potential of the omega-3 polyunsaturated fatty acid, eicosapentaenoic acid (EPA), which exerts anti-inflammatory effects. The results showed that the development of carcinogen-induced HCC was significantly less in mice fed a high-fat diet (HFD) supplemented with EPA than in those fed HFD only, suggesting that EPA attenuates the development of obesity-related HCC. Although EPA did not appear to affect obesity-linked inflammation, it suppressed the activation of the pro-tumorigenic IL-6 effector STAT3, contributing to the inhibition of tumor growth. These findings suggest a clinical implication of EPA as a treatment for obesity-related HCC.

Targeting the differential addiction to anti-apoptotic BCL-2 family for cancer therapy.

BCL-2 family proteins are central regulators of mitochondrial apoptosis and validated anti-cancer targets. Using small cell lung cancer (SCLC) as a model, we demonstrated the presence of differential addiction of cancer cells to anti-apoptotic BCL-2, BCL-XL or MCL-1, which correlated with the respective protein expression ratio. ABT-263 (navitoclax), a BCL-2/BCL-XL inhibitor, prevented BCL-XL from sequestering activator BH3-only molecules (BH3s) and BAX but not BAK. Consequently, ABT-263 failed to kill BCL-XL-addicted cells with low activator BH3s and BCL-XL overabundance conferred resistance to ABT-263. High-throughput screening identified anthracyclines including doxorubicin and CDK9 inhibitors including dinaciclib that synergized with ABT-263 through downregulation of MCL-1. As doxorubicin and dinaciclib also reduced BCL-XL, the combinations of BCL-2 inhibitor ABT-199 (venetoclax) with doxorubicin or dinaciclib provided effective therapeutic strategies for SCLC. Altogether, our study highlights the need for mechanism-guided targeting of anti-apoptotic BCL-2 proteins to effectively activate the mitochondrial cell death programme to kill cancer cells.

An interconnected hierarchical model of cell death regulation by the BCL-2 family.

Multidomain pro-apoptotic BAX and BAK, once activated, permeabilize mitochondria to trigger apoptosis, whereas anti-apoptotic BCL-2 members preserve mitochondrial integrity. The BH3-only molecules (BH3s) promote apoptosis by either activating BAX-BAK or inactivating anti-apoptotic members. Here, we present biochemical and genetic evidence that NOXA is a bona fide activator BH3. Using combinatorial gain-of-function and loss-of-function approaches in Bid(-/-)Bim(-/-)Puma(-/-)Noxa(-/-) and Bax(-/-)Bak(-/-) cells, we have constructed an interconnected hierarchical model that accommodates and explains how the intricate interplays between the BCL-2 members dictate cellular survival versus death. BID, BIM, PUMA and NOXA directly induce stepwise, bimodal activation of BAX-BAK. BCL-2, BCL-XL and MCL-1 inhibit both modes of BAX-BAK activation by sequestering activator BH3s and 'BH3-exposed' monomers of BAX-BAK, respectively. Furthermore, autoactivation of BAX and BAK can occur independently of activator BH3s through downregulation of BCL-2, BCL-XL and MCL-1. Our studies lay a foundation for targeting the BCL-2 family for treating diseases with dysregulated apoptosis.

Depletion of mitochondrial fission factor DRP1 causes increased apoptosis in human colon cancer cells.

Mitochondria play a critical role in regulation of apoptosis, a form of programmed cell death, by releasing apoptogenic factors including cytochrome c. Growing evidence suggests that dynamic changes in mitochondrial morphology are involved in cellular apoptotic response. However, whether DRP1-mediated mitochondrial fission is required for induction of apoptosis remains speculative. Here, we show that siRNA-mediated DRP1 knockdown promoted accumulation of elongated mitochondria in HCT116 and SW480 human colon cancer cells. Surprisingly, DRP1 down-regulation led to decreased proliferation and increased apoptosis of these cells. A higher rate of cytochrome c release and reductions in mitochondrial membrane potential were also revealed in DRP1-depleted cells. Taken together, our present findings suggest that mitochondrial fission factor DRP1 inhibits colon cancer cell apoptosis through the regulation of cytochrome c release and mitochondrial membrane integrity.