Chromomycins from soil-derived Streptomyces sp. inhibit the growth of human non-small cell lung cancer cells by targeting c-FLIP.

Three chromomycin derivatives, chromomycins A3 (1, CA3), A5 (2, CA5), and monodeacetylchromomycin A3 (3, MDA-CA3), were identified from the soil-derived Streptomyces sp. CGMCC 26516. A reinvestigation of the structure of CA5 is reported, of which the absolute configuration was unambiguously determined for the first time to be identical with that of CA3 based on nuclear magnetic resonance (NMR) data analysis as well as NMR and electronic circular dichroism calculations. Compounds 1-3 showed potent cytotoxicity against the non-small-cell lung cancer (NSCLC) cells (A549, H460, H157-c-FLIP, and H157-LacZ) and down-regulated the protein expression of c-FLIP in A549 cells. The IC50 values of chromomycins in H157-c-FLIP were higher than that in H157-LacZ. Furthermore, si-c-FLIP promoted anti-proliferation effect of chromomycins in NSCLC cells. In nude mice xenograft model, 1 and 2 both showed more potent inhibition on the growth of H157-lacZ xenografts than that of H157-c-FLIP xenografts. These results verify that c-FLIP mediates the anticancer effects of chromomycins in NSCLC.

Effective glioblastoma immune sonodynamic treatment mediated by macrophage cell membrane cloaked biomimetic nanomedicines.

Glioblastoma (GBM) as one of the most lethal brain tumours, remains poor therapeutic index due to its typical characters including heterogeneous, severe immune suppression as well as the existence of blood brain barrier (BBB). Immune sonodynamic (ISD) therapy combines noninvasive sonodynamic therapy with immunotherapy, which has great prospects for the combinational treatment of GBM. Herein, we develop macrophage cell membrane cloaked reactive oxygen species (ROS) responsive biomimetic nanoparticles, co-delivering of sonosensitizer Ce6 and JQ1 (a bromo-domain protein 4 (BRD4) inhibitor which can down-regulate PD-L1) and realizing potent GBM ISD therapy. The ApoE peptide decorated macrophage membrane coating endows these biomimetic nanoparticles with low immunogenicity, efficient BBB permeability, prolonged blood circulation half-live and good biocompatibility. The ROS responsive polymeric inner core could be readily degraded as triggered by excessive ROS under the ultrasound once they accumulated in tumour cells, fast release encapsulated drugs. The generation of ROS not only killed tumour cells via sonodynamic therapy, but also induced immunogenic cell death (ICD) and further activated the anti-tumour immune response. The released JQ1 inhibited tumour cell proliferation and augmented the immune activities by inhibiting the PD-L1 expression on the surface of tumour cells. The cascade sonodynamic and immune therapy resulted in significantly improved median survival time in both orthotopic GL261 and PTEN deficient immunosuppressive CT2A GBM mice models. Therefore, our developed biomimetic nanoparticle platform provides a promising combinational therapy strategy to treat immune suppressive GBM.

Thiostrepton suppresses triple-negative breast cancer through downregulating c-FLIP/SMAD2/3 signaling pathway.

Triple-negative breast cancer (TNBC) is an aggressive subtype with poor prognosis of breast cancer. Thiostrepton exerts anti-tumor activities against several cancers including TNBC. Herein we discussed the new molecular mechanisms of thiostrepton in TNBC. Thiostrepton inhibited MDA-MB-231 cell viability, accompanied by a decrease of c-FLIP and p-SMAD2/3. c-FLIP overexpression reduced the sensitivity of MDA-MB-231 cells to thiostrepton, while SMAD2/3 knockdown increased the sensitivity of MDA-MB-231 cells to thiostrepton. Moreover, c-FLIP overexpression significantly increased the expression and phosphorylation of SMAD2/3 proteins and vice versa. In conclusion, our study reveals c-FLIP/SMAD2/3 signaling pathway as a novel mechanism of antitumor activity of thiostrepton.

FASN Inhibitors Enhance Bestatin-Related Tumor Cell Apoptosis Through Upregulating PEPT1.

BACKGROUND: Fatty acid synthase (FASN) is generally over-expressed in human tumor tissues and catalyzes de novo synthesis of fatty acids on which tumor cells depend. Bestatin, an inhibitor of aminopeptidase/CD13, is one of the dipeptide substrates for the human oligopeptide transporter 1 (PEPT1). OBJECTIVES: In the current study, we aimed to uncover the role of FASN inhibitors in bestatininduced tumor cell apoptosis and the underlying mechanism, extending our understanding of the correlations between FASN and PEPT1 in cancer and providing a new strategy for tumor targeted treatment. METHODS: Cerulenin, orlistat and siRNAs were applied to inhibit FASN. The cell viability and apoptosis were assessed with MTT (thiazolyl blue tetrazolium bromide) assays and annexin VFITC/ PI staining with flow cytometry analysis. Western blot and qRT-PCR analysis were used to detect the protein levels and mRNA levels of the indicated genes in tumor cells, respectively. Protein degradation or stability was examined with cycloheximide chase assays. CD13 activity was detected by gelatin zymography. The HT1080 and C26 xenografts models were conducted to assess the efficacy in vivo. RESULTS: In the current study, we found that inhibiting FASN by cerulenin and orlistat both augmented the effects of bestatin in decreasing tumor cell viability. Cerulenin increased the apoptosis rates and enhanced the cleavage of PARP caused by bestatin. Furthermore, cerulenin, orlistat and siFASNs markedly elevated PEPT1 protein levels. Indeed, cerulenin induced the upregulation of PEPT1 mRNA expression rather than affecting the protein level after the cells were treated with CHX. And Gly-Sar, a typical competitive substrate of PEPT1, could attenuate the augment of bestatin-induced cell killing by cerulenin. Moreover, synergistic restrain of tumor growth accompanied by a reduction of Ki-67 and increment of TUNEL was significantly achieved in the xenograft models. Interestingly, no clear correlation was observed between the CD13 with FASN and/or PEPT1 in tumor cells. CONCLUSION: FASN inhibitors facilitate tumor cells susceptible to bestatin-induced apoptosis involving the up-regulation of PEPT1 at the mRNA translation level and the transport of bestatin by PEPT1, emerging as a promising strategy for tumor targeted therapy.

Thymidine Kinase 1 Mediates the Synergistic Antitumor Activity of Ubenimex and Celecoxib via Regulation of Cell Cycle in Colorectal Cancer.

Colorectal cancer (CRC) is a common clinical malignant tumor of the digestive system that seriously affects the health and life of patients. Because it is difficult to cure CRC, the strategy of drug combination is often used in clinical therapy. This study mainly revealed that ubenimex and/or celecoxib exerted anti-colon cancer effects in vitro and in vivo, and the efficacy was significantly enhanced when the two drugs were combined. The combination of the two drugs induced significantly stronger cell-cycle arrest than did the single drug, and also enhanced the antitumor efficacy of 5-fluorouracil and its derivatives. At the same time, the expression of thymidine kinase 1 (TK1) protein was decreased through regulating the level of TK1 mRNA treated with celecoxib and/or ubenimex, but the combination drugs exhibited much more reduction of TK1 mRNA and protein as compared with the single agent alone. TK1 may be the molecular target of the combination of two drugs to exert the anti-colorectal cancer effect. In summary, this research demonstrates that celecoxib combined with ubenimex inhibits the development of colorectal cancer in vitro and in vivo, making them a viable combination regimen. SIGNIFICANCE STATEMENT: In this study, our data reveal the great potential of celecoxib combined with ubenimex in the treatment of colorectal cancer, providing new ideas for clinical antitumor drug regimens and theoretical reference for drug development.

Brown adipocytes promote epithelial mesenchymal transition of neuroblastoma cells by inducing PPAR-γ/UCP2 expression.

Neuroblastoma (NB) is an embryonic malignant tumour of the sympathetic nervous system, and current research shows that activation of brown adipose tissue accelerates cachexia in cancer patients. However, the interaction between brown adipose tissues and NB remains unclear. The study aimed to investigate the effect of brown adipocytes in the co-culture system on the proliferation and migration of NB cells. Brown adipocytes promoted the proliferation and migration of Neuro-2a, BE(2)-M17, and SH-SY5Y cells under the co-culture system, with an increase of the mRNA and protein levels of UCP2 and PPAR-γ in NB cells. The UCP2 inhibitor genipin or PPAR-γ inhibitor T0090709 inhibited the migration of NB cells induced by brown adipocytes. Genipin or siUCP2 upregulated the expression of E-cadherin, and downregulated the expression of N-cadherin and vimentin in NB cells. We suggest that under co-cultivation conditions, NB cells can activate brown adipocytes, which triggers changes in various genes and promotes the proliferation and migration of NB cells. The PPAR-γ/UCP2 pathway is involved in the migration of NB cells caused by brown adipocytes.

c-FLIP promotes drug resistance in non-small-cell lung cancer cells via upregulating FoxM1 expression.

The forkhead box M1 (FoxM1) protein, a transcription factor, plays critical roles in regulating tumor growth and drug resistance, while cellular FLICE-inhibitory protein (c-FLIP), an anti-apoptotic regulator, is involved in the ubiquitin-proteasome pathway. In this study, we investigated the effects of c-FLIP on the expression and ubiquitination levels of FoxM1 along with drug susceptibility in non-small-cell lung cancer (NSCLC) cells. We first showed that the expression levels of FoxM1 and c-FLIP were increased and positively correlated (R2 = 0.1106, P < 0.0001) in 90 NSCLC samples. The survival data from prognostic analysis demonstrated that high expression of c-FLIP and/or FoxM1 was related to poor prognosis in NSCLC patients and that the combination of FoxM1 and c-FLIP could be a more precise prognostic biomarker than either alone. Then, we explored the functions of c-FLIP/FoxM1 in drug resistance in NSCLC cell lines and a xenograft mouse model in vivo. We showed that c-FLIP stabilized FoxM1 by inhibiting its ubiquitination, thus upregulated the expression of FoxM1 at post-transcriptional level. In addition, a positive feedback loop composed of FoxM1, ß-catenin and p65 also participated in c-FLIP-FoxM1 axis. We revealed that c-FLIP promoted the resistance of NSCLC cells to thiostrepton and osimertinib by upregulating FoxM1. Taken together, these results reveal a new mechanism by which c-FLIP regulates FoxM1 and the function of this interaction in the development of thiostrepton and osimertinib resistance. This study provides experimental evidence for the potential therapeutic benefit of targeting the c-FLIP-FoxM1 axis for lung cancer treatment.

Brain co-delivery of first-line chemotherapy drug and epigenetic bromodomain inhibitor for multidimensional enhanced synergistic glioblastoma therapy.

Glioblastoma (GBM) is a central nervous system tumor with poor prognosis due to the rapid development of resistance to mono chemotherapy and poor brain targeted delivery. Chemoimmunotherapy (CIT) combines chemotherapy drugs with activators of innate immunity that hold great promise for GBM synergistic therapy. Herein, we chose temozolomide, TMZ, and the epigenetic bromodomain inhibitor, OTX015, and further co-encapsulated them within our well-established erythrocyte membrane camouflaged nanoparticle to yield ApoE peptide decorated biomimetic nanomedicine (ABNM@TMZ/OTX). Our nanoplatform successfully addressed the limitations in brain-targeted drug co-delivery, and simultaneously achieved multidimensional enhanced GBM synergistic CIT. In mice bearing orthotopic GL261 GBM, treatment with ABNM@TMZ/OTX resulted in marked tumor inhibition and greatly extended survival time with little side effects. The pronounced GBM treatment efficacy can be ascribed to three key factors: (i) improved nanoparticle-mediated GBM targeting delivery of therapeutic agents by greatly enhanced blood circulation time and blood-brain barrier penetration; (ii) inhibited cellular DNA repair and enhanced TMZ sensitivity to tumor cells; (iii) enhanced anti-tumor immune responses by inducing immunogenic cell death and inhibiting PD-1/PD-L1 conjugation leading to enhanced expression of CD4+ and CD8+ T cells. The study validated a biomimetic nanomedicine to yield a potential new treatment for GBM.

Effect of solubilizing strategies on oral absorption of felodipine / 中国药科大学学报

@#As a typical BCS Ⅱ drug, felodipine exhibits low solubility and high permeability. We herein investigated the effects of different solubilization strategies on the oral absorption of felodipine. Firstly felodipine tablets based on 200 μm, 150 μm and 25 μm particle size of bulk drug were prepared. Meanwhile, felodipine solid dispersion and felodipine nanosuspension with average particle size of (168.90 ± 6.22) nm, PDI of 0.11 ± 0.06 were prepared. The absorption rate, apparent permeability coefficient (Papp), absorption quality in duodenum, jejunum, ileum and colon of rats and in vivo pharmacokinetics of the above different felodipine preparations were investigated. The results of rat single-pass intestinal perfusion showed that the absorption of felodipine preparations in duodenum, jejunum and ileum was better than in colon. Felodipine had a wide absorption window in the small intestine, with the best absorption site in the small intestine. Papp of different felodipine preparations was greater than 2.0 × 10-5 cm/s. Thus, the low solubility was the main factor limiting the absorption. In vivo pharmacokinetic experiments demonstrated the solubilization strategies significantly improved the bioavailability. The bioavailabilities of felodipine tablets with particle sizes of 150 and 25 μm, as well as nanosuspension, and solid dispersion were 138.75%, 173.01%, 208.65% and 314.53% that of the tablets with particle size of 200 μm, respectively. Solubilization strategies can significantly improve the gastrointestinal absorption rate and absorption quality of felodipine, and thus improve its bioavailability, which provides some reference for the research on the improvement of oral absorption of BCS II drugs.

Levamisole enhances DR4-independent apoptosis induced by TRAIL through inhibiting the activation of JNK in lung cancer.

THE HEADINGS AIMS: Levamisole has anti-parasite and antitumor activities, but the anti-lung cancer mechanism has not been studied. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is regarded as a promising drug because of the ability to selectively target cancer cells. However, the tolerance of cancer cells to TRAIL limits its antitumor activity. Other drugs combined with TRAIL need to be explored to enhance its antitumor activity. Based on the adjuvant anticancer effect of levamisole on anticancer drugs activity, the antitumor activity of levamisole combined with TRAIL will be investigated. MATERIALS AND METHODS: In vitro and in vivo experiments were employed to investigate the anti-tumor activity. Flow-cytometry analysis, western blotting and siRNA transfection were used to explore the molecular mechanism. KEY FINDINGS: Levamisole decreased the proliferation of lung cancer cells in vitro and in vivo and induced cell cycle arrest in G0/G1 phase. Besides, levamisole also enhanced TRAIL-induced DR4-independent apoptosis by inhibiting the phosphorylation of cJUN. A new cellular protective pathway LC3B-DR4/Erk was also disclosed, in which levamisole only increased the expression of LC3B and then activated the phosphorylation of Erk and increased the expression of DR4, while p-Erk and DR4 inter-regulated. SIGNIFICANCE: Levamisole may be used as an adjuvant of TRAIL in treating lung cancer. The discovery of LC3B-DR4/Erk as a new protective pathway provides a new direction for sensitizing lung cancer cells to TRAIL.

E3 ubiquitin ligase RNF170 inhibits innate immune responses by targeting and degrading TLR3 in murine cells.

Upon recognition of dsRNA, toll-like receptor 3 (TLR3) recruits the adaptor protein TRIF to activate IRF3 and NF-κB signaling, initiating innate immune responses. The ubiquitination of TLR3 downstream signaling molecules and their roles in the innate response have been discovered; however, whether TLR3 itself is ubiquitinated and then functionally involved remains to be elucidated. By immunoprecipitating TLR3-binding proteins in macrophages, we identified ring finger protein 170 (RNF170) as a TLR3-binding E3 ligase. RNF170 mediated the K48-linked polyubiquitination of K766 in the TIR domain of TLR3 and promoted the degradation of TLR3 through the proteasome pathway. The genetic ablation of RNF170 selectively augmented TLR3-triggered innate immune responses both in vitro and in vivo. Our results reveal a novel role for RNF170 in selectively inhibiting TLR3-triggered innate immune responses by promoting TLR3 degradation.

Honokiol inhibits breast cancer cell metastasis by blocking EMT through modulation of Snail/Slug protein translation.

Honokiol (HNK), an active compound isolated from traditional Chinese medicine Magnolia officinalis, has shown potent anticancer activities. In the present study, we investigated the effects of HNK on breast cancer metastasis in vitro and in vivo, as well as the underlying molecular mechanisms. We showed that HNK (10-70 µmol/L) dose-dependently inhibited the viability of human mammary epithelial tumor cell lines MCF7, MDA-MB-231, and mouse mammary tumor cell line 4T1. In the transwell and scratch migration assays, HNK (10, 20, 30 µmol/L) dose-dependently suppressed the invasion and migration of the breast cancer cells. We demonstrated that HNK (10-50 µmol/L) dose-dependently upregulated the epithelial marker E-cadherin and downregulated the mesenchymal markers such as Snail, Slug, and vimentin at the protein level in breast cancer cells. Using a puromycin incorporation assay, we showed that HNK decreased the Snail translation efficiency in the breast cancer cells. In a mouse model of tumor metastasis, administration of HNK (50 mg/kg every day, intraperitoneal (i.p.), 6 times per week for 30 days) significantly decreased the number of metastatic 4T1 cell-derived nodules and ameliorated the histological alterations in the lungs. In addition, HNK-treated mice showed decreased Snail expression and increased E-cadherin expression in metastatic nodules. In conclusion, HNK inhibits EMT in the breast cancer cells by downregulating Snail and Slug protein expression at the mRNA translation level. HNK has potential as an integrative medicine for combating breast cancer by targeting EMT.

IMB0901 inhibits muscle atrophy induced by cancer cachexia through MSTN signaling pathway.

BACKGROUND: Cancer cachexia as a metabolic syndrome can lead to at least 25% of cancer deaths. The inhibition of muscle atrophy is a main strategy to treat cancer cachexia. In this process, myostatin (MSTN) can exert a dual effect on protein metabolism, including inhibition of protein biosynthesis and enhancement of protein degradation. In this study, we will test the effect on muscle atrophy induced by cancer cachexia of IMB0901, a MSTN inhibitor. METHODS: Two high-throughput screening models against MSTN were developed. By screening, IMB0901, 2-((1-(3,4-dichlorophenyl)-1H-pyrazolo [3,4-d] pyrimidin-4-yl) amino) butan-1-ol, was picked out from the compound library. The in vitro cell model and the C26 animal model of muscle atrophy induced by cancer cachexia were used to determine the pharmacological activity of IMB0901. Whether IMB0901 could inhibit the aggravating effect of doxorubicin on muscle wasting was examined in vitro and in vivo. RESULTS: IMB0901 inhibited the MSTN promoter activity, the MSTN signaling pathway, and the MSTN positive feedback regulation. In atrophied C2C12 myotubes, IMB0901 had a potent efficiency of decreasing MSTN expression and modulating MSTN signaling pathway which was activated by C26-conditioned medium (CM). In C2C12 myotubes, the expressions of three common myotube markers, myosin heavy chain (MyHC), myogenic differentiation 1 (MyoD), and myogenin (MyoG), were downregulated by CM, which could be efficiently reversed by IMB0901 via reduction of ubiquitin-mediated proteolysis and enhancement of AKT/mTOR-mediated protein synthesis. In the C26 animal model, IMB0901 mitigated the weight loss of body, quadricep and liver, and protected the quadriceps cell morphology. Furthermore, IMB0901 decreased the expression of two E3 ligases Atrogin-1 and MuRF-1 in the quadriceps in vivo. At the cellular level, IMB0901 had no influence on anti-tumor effect of three chemotherapeutic agents (cisplatin, doxorubicin, and gemcitabine) and lowered doxorubicin-induced upregulation of MSTN in C2C12 myotubes. IMB0901 did not affect the inhibitory effect of doxorubicin on C26 tumor and delayed the weight loss of muscle and adipose tissue caused by C26 tumor and doxorubicin. CONCLUSIONS: IMB0901 inhibits muscle atrophy induced by cancer cachexia by suppressing ubiquitin-mediated proteolysis and promoting protein synthesis. These findings collectively suggest that IMB0901 is a promising leading compound for the management of muscle atrophy induced by cancer cachexia.

Effect of brown adipose tissue/cells on the growth of mouse hepatocellular carcinoma in vitro and in vivo.

Activation of brown adipose tissue (BAT) is an effective strategy for treating obesity. Hepatocellular carcinoma (HCC) is a life-threatening hepatic malignancy with a high mortality rate. Considering that obesity is a risk factor for HCC, the aim of the present study was to investigate the association between HCC and BAT. Using a mouse model, H22 transplantation led to an increase in liver weight, a decrease in the weight of BAT and white adipose tissue, and an increase in the serum level of triacylglycerol (TG). In the in vivo BAT excision model, the removal of BAT led to increased growth of H22 tumors, which was accompanied by a more marked increase in liver weight and in the serum level of TG. The in vitro and in vivo intervention models with primary brown adipose cells (BACs) indicated that primary BACs can directly decrease the viability of H22 cells and the growth of tumors. In conclusion, BAT is a protective organ or tissue against HCC, and BACs may be a potential therapeutic tool for the treatment of HCC.

Self-Recognition of an Inducible Host lncRNA by RIG-I Feedback Restricts Innate Immune Response.

The innate RNA sensor RIG-I is critical in the initiation of antiviral type I interferons (IFNs) production upon recognition of "non-self" viral RNAs. Here, we identify a host-derived, IFN-inducible long noncoding RNA, lnc-Lsm3b, that can compete with viral RNAs in the binding of RIG-I monomers and feedback inactivate the RIG-I innate function at late stage of innate response. Mechanistically, binding of lnc-Lsm3b restricts RIG-I protein's conformational shift and prevents downstream signaling, thereby terminating type I IFNs production. Multivalent structural motifs and long-stem structure are critical features of lnc-Lsm3b for RIG-I binding and inhibition. These data reveal a non-canonical self-recognition mode in the regulation of immune response and demonstrate an important role of an inducible "self" lncRNA acting as a potent molecular decoy actively saturating RIG-I binding sites to restrict the duration of "non-self" RNA-induced innate immune response and maintaining immune homeostasis, with potential utility in inflammatory disease management.

Nuclear RNF2 inhibits interferon function by promoting K33-linked STAT1 disassociation from DNA.

Prolonged activation of interferon-STAT1 signaling is closely related to inflammatory autoimmune disorders, and therefore the identification of negative regulators of these pathways is important. Through high-content screening of 115 mouse RING-domain E3 ligases, we identified the E3 ubiquitin ligase RNF2 as a potent inhibitor of interferon-dependent antiviral responses. RNF2 deficiency substantially enhanced interferon-stimulated gene (ISG) expression and antiviral responses. Mechanistically, nuclear RNF2 directly bound to STAT1 after interferon stimulation and increased K33-linked polyubiquitination of the DNA-binding domain of STAT1 at position K379, in addition to promoting the disassociation of STAT1/STAT2 from DNA and consequently suppressing ISG transcription. Our study provides insight into the regulation of interferon-dependent responses via a previously unrecognized post-translational modification of STAT1 in the nucleus.

RNF122 suppresses antiviral type I interferon production by targeting RIG-I CARDs to mediate RIG-I degradation.

The activation of retinoic acid-inducible gene 1 (RIG-I), a cytoplasmic innate sensor for viral RNA, is tightly regulated to maintain immune homeostasis properly and prevent excessive inflammatory reactions other than initiation of antiviral innate response to eliminate RNA virus effectively. Posttranslational modifications, particularly ubiquitination, are crucial for regulation of RIG-I activity. Increasing evidence suggests that E3 ligases play important roles in various cellular processes, including cell proliferation and antiviral innate signaling. Here we identify that E3 ubiquitin ligase RING finger protein 122 (RNF122) directly interacts with mouse RIG-I through MS screening of RIG-I-interacting proteins in RNA virus-infected cells. The transmembrane domain of RNF122 associates with the caspase activation and recruitment domains (CARDs) of RIG-I; this interaction effectively triggers RING finger domain of RNF122 to deliver the Lys-48-linked ubiquitin to the Lys115 and Lys146 residues of RIG-I CARDs and promotes RIG-I degradation, resulting in a marked inhibition of RIG-I downstream signaling. RNF122 is widely expressed in various immune cells, with preferential expression in macrophages. Deficiency of RNF122 selectively increases RIG-I-triggered production of type I IFNs and proinflammatory cytokines in macrophages. RNF122-deficient mice exhibit more resistance against lethal RNA virus infection, with increased production of type I IFNs. Thus, we demonstrate that RNF122 acts as a selective negative regulator of RIG-I-triggered antiviral innate response by targeting CARDs of RIG-I and mediating proteasomal degradation of RIG-I. Our study outlines a way for E3 ligase to regulate innate sensor RIG-I for the control of antiviral innate immunity.

RACK1 Promotes Autophagy by Enhancing the Atg14L-Beclin 1-Vps34-Vps15 Complex Formation upon Phosphorylation by AMPK.

Autophagy is essential for maintaining tissue homeostasis. Although adaptors have been demonstrated to facilitate the assembly of the Atg14L-Beclin 1-Vps34-Vps15 complex, which functions in autophagosome formation, it remains unknown whether the autophagy machinery actively recruits such adaptors. WD40-repeat proteins are a large, highly conserved family of adaptors implicated in various cellular activities. However, the role of WD40-repeat-only proteins, such as RACK1, in postnatal mammalian physiology remains unknown. Here, we report that hepatocyte-specific RACK1 deficiency leads to lipid accumulation in the liver, accompanied by impaired Atg14L-linked Vps34 activity and autophagy. Further exploration indicates that RACK1 participates in the formation of autophagosome biogenesis complex upon its phosphorylation by AMPK at Thr50. Thr50 phosphorylation of RACK1 enhances its direct binding to Vps15, Atg14L, and Beclin 1, thereby promoting the assembly of the autophagy-initiation complex. These observations provide insight into autophagy induction and establish a pivotal role for RACK1 in postnatal mammalian physiology.

RACK1 expression contributes to JNK activity, but JNK activity does not enhance RACK1 expression in hepatocellular carcinoma SMMC-7721 cells.

Receptor for activated C kinase 1 (RACK1) is up-regulated in hepatocellular carcinoma (HCC) and has been reported to augment c-Jun N-terminal protein kinase (JNK) activity in HCC SMMC-7721 cells. By contrast, activator protein-1, a downstream JNK transcription factor, has been revealed to mediate the overexpression of RACK1 in melanoma cells. Therefore, the association between RACK1 and JNK activity in HCC cells has yet to be completely elucidated. The present study analyzed the effects of RACK1 or JNK loss of function on the levels of RACK1 protein, JNK activity, cell proliferation and apoptosis induced by tumor necrosis factor-related apoptosis inducing ligand in HCC SMMC-7721 cells. It was found that JNK loss of function exhibited no effect on RACK1 expression, whereas a loss of RACK1 function led to reduced JNK activity in SMMC-7721 cells. RACK1 and JNK loss of function resulted in the impaired oncogenic growth of SMMC-7721 cells. The present data further support a pivotal role of RACK1 in mediating enhanced JNK activity in HCC cells and also indicate that a novel mechanism exists for RACK1 overexpression in HCC SMMC-7721 cells.