Knockdown Of Beclin Research Articles

Abstract 4262: MiR-873 is the master regulator of autophagy genes through a novel negative feedback mechanism mediated by Elongation factor 2 kinase (eEF-2K) and suppresses tumor growth and progression of triple negative breast cancer

Abstract Autophagy is a highly complex lysosomal degradation process. Recently we demonstrated that autophagy genes encoding Beclin1, ATG7, LC3 promotes cell proliferation, survival, and invasion by promoting Cyclin D1 and Integrin β1/ Src signaling in triple negative breast cancer (TNBC). Studies showed that increased basal autophagy is linked to development of chemoresistance, relapses and metastasis and poor prognosis in TNBC. However, the major molecular mechanisms and the integrated global regulators controlling the autophagic machinery still remains unknown. Here, we identified miRNA-873 as a p53-driven tumor suppressor that is associated with favorable patient survival (TCGA database) and the key factor for regulating the major autophagy genes, including BCN1, LC3, ATG7, ATG16L1 and ATG13 in TNBC. Using in silico prediction algorithms we demonstrated that miR-873 has binding sites on the 3’-untranslated region (3’-UTR) of these genes and directly binds and suppresses their expression using by gene reporter assays. Introduction of mutations to the miR-873 binding sites on 3-UTR of these genes reversed the inhibitory effect of miR-873on these genes. Furthermore, knockdown of Beclin1, LC3 and ATG7 genes significantly suppressed Eukaryotic Elongation Factor-2 kinase (eEF2K), an unusual alpha kinase, which is highly overexpressed in TNBC patients and associated with poor prognosis. We also found that miR-873 also binds to the 3’-UTR of eEF2K mRNA and regulates its expression and inhibits starvation induced autophagy. Through knockdown and overexpression studies we also demonstrated that eEF2K regulates expression of abovementioned autophagic proteins. Interestingly, we found that BCN1, LC3, and ATG7 also regulates expression of EF2K, suggesting an existence of a novel negative feed-back loop. Lastly, we demonstrated that miR-873 expression is suppressed in TNBC patients and cell lines and restoration of its expression in vivo in MDA-MB-231 and MDA-MB-436 orthotopic xenograft models by systemic injection (I.V, tail vein once a week, 0.15 mg/kg) of miR-873 mimic molecules incorporated in novel single lipid (SLNP)-nanoparticles suppressed tumor growth. Furthermore, in vivo treatment of mice with SLNP-Beclin1, ATG7 or ATG8 siRNAs also completely suppressed TNBC tumor growth. In conclusion, our data suggest that p53/miR-873/eEF2K axis is a novel post-transcriptional regulator of autophagy and miR-873 functions as a master regulator of the post-transcriptional regulation of the major autophagy genes directly and indirectly through eEF2K dependent dual-suppressor mechanism and modulation of this axis could be used as a potential therapy for TNBC. Citation Format: Hamada Ahmed Mokhlis, Nermin Kahraman, Seyda Baydogan, Abdel-Aziz Hamed Abdel-Aziz, Ahmed Ashour, Cristina Ivan, Gabriel Lopez-Berestein, Bulent Ozpolat. MiR-873 is the master regulator of autophagy genes through a novel negative feedback mechanism mediated by Elongation factor 2 kinase (eEF-2K) and suppresses tumor growth and progression of triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4262.

Curcumin overcome primary gefitinib resistance in non-small-cell lung cancer cells through inducing autophagy-related cell death

BackgroundEpidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) are being wildly used as target therapy in non-small-cell lung cancer (NSCLC). However, NSCLC patients with wild-type EGFR and KRAS mutation are primary resistant to EGFR-TKIs such as gefitinib. Curcumin has been known as a potential therapeutic agent for several major human cancers. In this study, we investigated the effect of curcumin on the reversal of gefitinib resistance in NSCLC cells as well as their molecular bases.MethodsH157 (wild-type EGFR and KARS mutation) and H1299 (wild-type EGFR and HRAS mutation) cells were treated with gefitinib or curcumin alone, or the two combination, and then cell viability, EGFR activity, expressions of Sp1 and Sp1-dependent proteins and receptor tyrosine kinases, markers of autophagy and apoptosis were examined by using CCK-8, colony formation, immunoblot, quantitative PCR, immunofluoscence, and flow cytometry assays. Also xenograft experiments were conduced to test the synergism of curcumin to gefitinib.ResultsOur results showed that curcumin significantly enhanced inhibitory effect of gefitinib on primary gefitinib-resistant NSCLC cell lines H157 and H1299. Combination treatment with curcumin and gefitinib markedly downregulated EGFR activity through suppressing Sp1 and blocking interaction of Sp1 and HADC1, and markedly suppressed receptor tyrosine kinases as well as ERK/MEK and AKT/S6K pathways in the resistant NSCLC cells. Meanwhile, combination treatment of curcumin and gefitinib caused dramatic autophagy induction, autophagic cell death and autophagy-mediated apoptosis, compared to curcumin or gefitinib treatment alone, as evidenced by the findings that curcumin and gefitinib combination treatment-produced synergistic growth inhibition and apoptosis activation can be reversed by pharmacological autophagy inhibitors (Baf A1 or 3-MA) or knockdown of Beclin-1 or ATG7, also can be partially returned by pan-caspase inhibitor (Z-VAD-FMK) in H157 and H1299 cells. Xenograft experiments in vivo yielded similar results.ConclusionsThese data indicate that the synergism of curcumin on gefitinib was autophagy dependent. Curcumin can be used as a sensitizer to enhance the efficacy of EGFR-TKIs and overcome the EGFR-TKI resistance in NSCLC patients with wild-type EGFR and/or KRAS mutation.

Inositol-requiring enzyme 1 alpha endoribonuclease specific inhibitor STF-083010 protects the liver from thioacetamide-induced oxidative stress, inflammation and injury by triggering hepatocyte autophagy

Acute liver injury caused by toxins or drugs is a common condition that threatens patients' lives. Inositol-requiring enzyme 1 alpha (IRE1α), the most conserved endoplasmic reticulum (ER) stress sensor, has been implicated in the pathophysiology of liver injury. Activated IRE1α endoribonuclease (RNase) can splice X-box binding protein 1 (XBP1) mRNA to produce the sXBP1 transcription factor. STF-083010, a specific inhibitor of IRE1α RNase, has recently been suggested to exhibit anti-oxidant and anti-inflammatory properties in multiple injury models. However, it remains unknown whether STF-083010 has a protective effect against thioacetamide (TAA)-induced acute liver injury. Here, we demonstrated that IRE1α-sXBP1 signaling is involved in the development of TAA-induced acute liver injury and correlates with the severity of liver damage. STF-083010 protected against TAA-induced liver injury, as evidenced by higher survival rates in response to a lethal dose of TAA and less severe liver injury in response to a toxic dose of TAA. Mechanistic exploration showed that STF-083010 triggered hepatocyte autophagy in response to TAA stimulation both in vivo and in vitro, leading to reduced reactive oxygen species (ROS) production and attenuated hepatic inflammation. We also found that Beclin-1 played a critical role in STF-083010-mediated autophagy in response to TAA stimulation. Autophagy inhibition by chloroquine (CQ) in vivo and Beclin-1 knockdown in vitro markedly abrogated the protective role of STF-083010 against TAA-induced oxidative stress, inflammation and hepatotoxicity. Our results suggested STF-083010 as a potential therapeutic application to prevent TAA-induced acute liver injury.

Critical role of autophagy regulator Beclin1 in endothelial cell inflammation and barrier disruption

Recent studies have implicated autophagy in several inflammatory diseases involving aberrant endothelial cell (EC) responses, such as acute lung injury (ALI). However, the mechanistic basis for a role of autophagy in EC inflammation and permeability remain poorly understood. In this study, we impaired autophagy by silencing the essential Beclin1 autophagy gene in human pulmonary artery EC. This resulted in reduced expression of proinflammatory genes in response to thrombin, a procoagulant and proinflammatory mediator whose concentration is elevated in many diseases including sepsis and ALI. These (Beclin1-depleted) cells also displayed a marked decrease in NF-κB activity secondary to impaired DNA binding of RelA/p65 in the nucleus, but exhibited normal IκBα degradation in the cytosol. Further analysis showed that Beclin1 knockdown was associated with impaired RelA/p65 translocation to the nucleus. Additionally, Beclin1 knockdown attenuated thrombin-induced phosphorylation of RelA/p65 at Ser536, a critical event necessary for the transcriptional activity of RelA/p65. Beclin1 silencing also protected against thrombin-induced EC barrier disruption by preventing the loss of VE-cadherin at adherens junctions. Moreover, Beclin1 knockdown reduced thrombin-induced phosphorylation/inactivation of actin depolymerizing protein Cofilin1 and thereby actin stress fiber formation required for EC permeability as well as RelA/p65 nuclear translocation. Together, these data identify Beclin1 as a novel mechanistic link between autophagy and EC dysfunction (inflammation and permeability).

Ox-LDL-induced lncRNA MALAT1 promotes autophagy in human umbilical vein endothelial cells by sponging miR-216a-5p and regulating Beclin-1 expression

Metastasis associated lung adenocarcinoma transcript 1 (MALAT1) has been validated as a potent protective agent of Atherosclerosis (AS). Here, we explored the molecular mechanism of MALAT1 exerting protective function in oxidized low-density lipoprotein (ox-LDL)-stimulated human umbilical vein endothelial cells (HUVECs). qRT-PCR assay was used to assess the expression of MALAT1 and miR-216a-5p. Western blot analysis was performed to detect LC3-I, LC3-II, Beclin-1 and p62 levels. The autophagosome formation was analyzed by immunofluorescence analysis. Cell apoptosis was measured by flow cytemotry and caspase 3 activity. Dual-luciferase reporter assay or RNA immunorecipitation assay was performed to verify the targeted interrelation between MALAT1 and miR-216a-5p, or miR-216a-5p and Beclin-1. Our data revealed that MALAT1 was upregulated and miR-216a-5p was downregulated in serum of AS patients and ox-LDL-treated HUVECs. ox-LDL treatment induced HUNECs autophagy. Moreover, MALAT1 enhanced autophagy and survival in ox-LDL-treated HUVECs. MALAT1 directly binded to miR-216a-5p and MALAT1 enhanced Beclin-1 expression by sponging miR-216a-5p. Also, miR-216-5p-mediated repressive effect on autophagy and survival was abrogated by MALAT1. Additionally, Beclin-1 knockdown inhibited autophagy and survival in ox-LDL-treated HUVECs. In all, our data suggested that MALAT1 might play a protective role at least partly by sponging miR-216a-5p and regulating Beclin-1, highlighting that MALAT1 might be a potential therapeutic target of AS.

The mechanism of Demethy1zeylasteral (ZST93) on autophagy in human pancreatic cancer cells

Objective To explore the mechanism of the novel tripterygium wilfordii hook F(TWHF) extract Demethylzeylasteral (ZST93) on autophagy of human pancreatic cancer (PC) cells. Methods Autophagy levels induced by ZST93 were evaluated by immunofluorescence and transmission electron microscopy in human pancreatic cancer cell lines. The activity levels of extracellular signal-regulated kinase1/2 (ERK1/2) and protein kinase B/mammalian target of rapamycin (Akt/mTOR) pathways were determined by Western blot in PC cells treated with different concentrations of ZST93. Immunofluorescence and transmission electron microscopy were used to evaluate the autophagy levels induced by ZST93 after inhibition of ERK1/2 pathway with PD 98059. Results Low concentrations of ZST93 could induce autophagy in PC cells. The LC3 expression were elevated to 3.4 and 19 times to control groups in MIA PaCa-2 and PANC-1 cells, respectively. And the number of autophagic vacuole were increased to 13 and 7 times to control groups, respectively. The knock-down of Beclin1 effectively inhibited the autophagy process induced by ZST93, with that LC3 expression reduced to 1.9 (P<0.05) and 2 (P<0.01) times to control groups in MIA PaCa-2 and PANC-1 cells, respectively, and the number of autophagic vacuole reduced to 2 (P<0.01) and 1 time (P<0.01) to control groups, respectively. The autophagy induced by low concentrations of ZST93 on PC cells was associated with the significant up-regulation of phospho-ERK1/2 (p-ERK1/2) and down-regulation of phospho-Akt (p-Akt) and phospho-mTOR (p-mTOR) expression. After introduction of low concentrations of ZST93, p-ERK/ERK was increased from 1.03±0.11 to 1.84±0.13 (P<0.01), while p-Akt/Akt and p-mTOR/mTOR were reduced from 0.98±0.07 to 0.51±0.05 (P<0.01) and from 1.00±0.03 to 0.63±0.02 (P<0.01), respectively, in MIA PaCa-2 cells. In PANC-1 cells, p-ERK/ERK was increased from 1.15±0.13 to 1.86±0.09 (P<0.01), while p-Akt/Akt and p-mTOR/mTOR were reduced from 1.02±0.06 to 0.43±0.05 (P<0.01) and from 0.99±0.02 to 0.61±0.01 (P<0.01), respectively. Treatment with high concentrations of ZST93 did not show such effects. Moreover, ERK1/2 pathway inhibitor PD 98059 could effectively inhibit the elevated level of autophagy induced by low-dose ZST93. Conclusion Low concentrations of ZST93 could induce Beclin1-dependent autophagy by activating ERK1/2 pathway and inhibiting Akt/mTOR pathway. Key words: Demethylzeylasteral; Autophagy; Pancreatic cancer; Extracellular signal-regulated kinase1/2 pathway; Protein kinase B/mammalian target of rapamycin pathway

Protective autophagy decreases osimertinib cytotoxicity through regulation of stem cell-like properties in lung cancer.

Osimertinib, a third-generation epidermal growth factor receptor - tyrosine kinase inhibitor (EGFR-TKI), shows great efficacy in EGFR-mutant non-small cell lung cancer (NSCLC); however, the resistance is inevitable. Osimertinib induces autophagy in NSCLC cells, but the role of autophagy in osimertinib resistance is not clear. We discovered that enhanced autophagy is associated with osimertinib resistance in vitro and in vivo. Inhibition of autophagy enhanced osimertinib cytotoxicity in both osimertinib-resistant and sensitive cells. Moreover, osimertinib-resistant cells exhibited stem cell-like properties, whereas autophagy inhibition decreased the stemness by downregulating the expression of SOX2 and ALDH1A1. Further, we found that knockdown of Beclin-1 inhibited the stem cell-like properties and restored osimertinib cytotoxicity. Osimertinib combined with chloroquine inhibited tumor growth more effectively than alone in xenograft mice. These results reveal that autophagy plays an adverse role in osimertinib cytotoxicity through inducing stem cell-like properties. Combination therapy of EGFR-TKI and autophagy inhibitor could provide a promising strategy to improve osimertinib cytotoxicity.

Osteosarcoma cell intrinsic PD-L2 signals promote invasion and metastasis via the RhoA-ROCK-LIMK2 and autophagy pathways

Known as co-stimulatory molecule, programmed death ligand-2 (PD-L2) contributes to T-cell exhaustion by interaction with programmed death-1 (PD-1) receptor, but its tumor cell-intrinsic signal effects have been little investigated. PD-L2 expression was detected by immunohistochemistry in 18 pairs of primary osteosarcoma tissues and matching lung metastasis tissues. We also investigated the effects of PD-L2 knockdown on osteosarcoma both in vitro and in vivo. In our study, PD-L2 expression was elevated in lung metastases compared with primary osteosarcoma according to an immunohistochemistry assay. Wound-healing and transwell assays revealed that PD-L2 knockdown leaded to inhibition of migration and invasion of human osteosarcoma cells in vitro. Mechanistically, we demonstrated that PD-L2 knockdown attenuated migration and invasion by inactivating RhoA-ROCK-LIMK2 signaling, suppressing epithelial–mesenchymal transition (EMT), and inhibiting autophagy by decreasing beclin-1 expression. In support of these observations, beclin-1 knockdown also inhibited activation of the RhoA-ROCK-LIMK2 pathway, leading to autophagy inhibition-induced blockade of migration and invasion. Depletion of PD-L2 in KHOS cells markedly weakens pulmonary metastatic potential in vivo by orthotopic transplantation of nude mice. Our study reveals a pro-metastatic functional mechanism for PD-L2 in osteosarcoma. Furthermore, we demonstrate a regulatory role for PD-L2 on autophagy, as well as a relationship between autophagy and metastasis in osteosarcoma, which may represent a potential therapeutic target for osteosarcoma.

Autophagy induction by xanthoangelol exhibits anti-metastatic activities in hepatocellular carcinoma.

Xanthoangelol (XAG), a prenylated chalcone isolated from the Japanese herb Angelica keiskei Koidzumi, has been reported to exhibit antineoplastic properties. However, the specific anti-tumor activity of XAG in human hepatocellular carcinoma (HCC), and the relevant mechanisms are not known. Herein, we evaluated the effect of XAG against HCC in vitro and in vivo. Although XAG treatment did not significantly reduce the viability of the Hep3B and Huh7 cell lines, it suppressed cell migration, invasion, and EMT. This anti-metastatic effect of XAG was due to induction of autophagy, because treatment with the autophagy inhibitor 3-methyadenine (3-MA) or knockdown of the pro-autophagy Beclin-1 effectively abrogated the XAG-induced suppression of metastasis. Mechanistically, XAG induced autophagy via activation of the AMPK/mTOR signaling pathway, and XAG treatment dramatically increased the expression of p-AMPK while decreasing p-mTOR expression. In addition, blocking AMPK/mTOR axis with compound C abrogated the autophagy-mediated inhibition of metastasis. The murine model of HCC metastasis also showed that XAG effectively reduced the number of metastatic pulmonary nodules. Taken together, our results revealed that autophagy via the activation of AMPK/mTOR pathway is essential for the anti-metastatic effect of XAG against HCC. These findings not only contribute to our understanding of the anti-tumor activity of XAG but also provide a basis for its clinical application in HCC. Before this study, evidence of XAG on HCC was purely anecdotal; present study provides the first comprehensive assessments of XAG on HCC metastasis and investigates its underlying mechanism. Results suggest that XAG exerts anti-metastatic properties against HCC through inducing autophagy which is mediated by the activation of AMPK/mTOR signaling pathway. This research extends our knowledge about the antineoplastic properties of XAG and suggests that induction autophagy may represent future treatment strategies for metastatic HCC.

HepatitisC virus nonstructural5A protein inhibits the starvation‑induced apoptosis of hepatoblastoma cells by increasing Beclin1 expression.

Hepatitis C virus (HCV) nonstructural protein 5A (NS5A) modulates cellular apoptosis, which is involved in the occurrence and development of liver cancer. The mechanisms of apoptosis inhibition by NS5A in liver cancer cells remains unclear. Beclin 1, which functions upstream of autophagosome formation, is upregulated by NS5A. Autophagy, an evolutionarily conserved catabolic process, has a crucial role in tumor initiation and progression. Autophagy was blocked by inhibitors 3‑methyladenine and chloroquine, or via knockdown of Beclin 1. Flow cytometric analysis and western blotting were used to detect apoptosis. It was found that inhibition of autophagy attenuated the NS5A‑mediated apoptosis inhibition of HepG2 cells. Furthermore, it was confirmed that Beclin 1 expression by NS5A was involved in the negative regulation of starvation‑induced liver cancer apoptosis, which was accompanied by reduced p53 and apoptosis regulator Bax expression, as well as decreased caspase‑3/-7 activation. Therefore, inhibition of autophagy may be promising therapeutic strategy in the treatment of HCV‑associated liver cancer.

Effect of Beclin-1 shRNA on hypoxia-induced autophagy in SH-SY5Y cells

To observe the effect of hypoxia on autophagy in Beclin-1-knockdown SH-SY5Y cells by constructing a stable transfected SH-SY5Y cell lines of silencing Beclin-1 gene. Beclin-1shRNA lentiviral vector and negative control lentiviral vector were constructed; the vector was transfected into SH-SY5Y cells; then the expression of Beclin-1 mRNA was detected by RT-PCR, the level of Beclin-1 protein was detected by Western blot. CCK-8 method was used to determine the effect of Beclin-1 knockdown on the viability of SH-SY5Y cells. Next, the blank control, negative control and transfected cells were cultured under 21% normoxia and 5% hypoxia conditions. The expression of LC3 protein in each group was detected by Western blot and the autophagic bodies were observed by electron microscopy. Beclin-1 shRNA significantly inhibited the expression of Beclin-1 mRNA and protein in SH-SY5Y cells; after silencing Beclin 1 gene, the survival rate of Beclin-1 shRNA group cells was no different from that of negative control (NC) group. After 5% hypoxia treatment, compared with NC group, the ratio of LC3Ⅱ/LC3Ⅰand the number of autophagy bodies were all decreased in Beclin-1 shRNA group. Beclin-1 knockdown SH-SY5Y cell lines and negative control cell lines were successfully established. Lentivirus-mediated Beclin-1 shRNA has no effect on the viability of SH-SY5Y cells, but can inhibit hypoxia-induced autophagy.

Beclin1 inhibition enhances paclitaxel‑mediated cytotoxicity in breast cancer invitro and invivo.

Beclin1, a key regulator of autophagy, has been demonstrated to be associated with cancer cell resistance to chemotherapy. Paclitaxel is a conventional chemotherapeutic drug used in the clinical treatment of breast cancer. However, the function and mechanism of Beclin1 in paclitaxel‑mediated cytotoxicity in breast cancer are not well defined. The present study demonstrated that paclitaxel suppressed cell viability and Beclin1 expression levels in BT‑474 breast cancer cells in a dose‑ and time‑dependent fashion. Compared with the control, the knockdown of Beclin1 significantly enhanced breast cancer cell death via the induction of caspase‑dependent apoptosis following paclitaxel treatment in vitro (P<0.05). In a BT‑474 xenograft model, paclitaxel achieved substantial inhibition of tumor growth in the Beclin1 knockdown group compared with the control group. Furthermore, analysis of the publicly available Gene Expression Omnibus datasets revealed a clinical correlation between Beclin1 levels and the response to paclitaxel therapy in patients with breast cancer. Collectively, the present results suggest that Beclin1 protects breast cancer cells from apoptotic death. Thus, the inhibition of Beclin1 may be a novel way to improve the effect of paclitaxel. Additionally, Beclin1 may function as a favorable prognostic biomarker for paclitaxel treatment in patients with breast cancer.

PI3K/Akt-Beclin1 signaling pathway positively regulates phagocytosis and negatively mediates NF-κB-dependent inflammation in Staphylococcus aureus-infected macrophages

Although autophagy and phagocytosis are involved in the regulation of host inflammatory response to bacterial infection in macrophages, the underlying mechanisms have not been completely elucidated. In the present study, we found that infecting RAW264.7 macrophages with Staphylococcus aureus (S. aureus) activated multiple signaling pathways including phosphatidylinositide 3-kinase (PI3K)/protein kinase B (Akt), nuclear factor-κB (NF-κB), and Rac1, as well as triggered autophagy. LY294002, a specific PI3K activity inhibitor, significantly decreased autophagy and phagocytosis of macrophages upon S. aureus infection. Similarly, knockdown of Beclin1 by specific siRNA significantly inhibited autophagy and phagocytosis of S. aureus-infected macrophages. Additionally, we showed that although administration of Beclin1 siRNA had no effects on phosphorylation of Akt (p-Akt), inhibition of PI3K activity by LY294002 significantly decreased the expression of Beclin1, suggesting that Beclin1 is a downstream molecular of PI3K. Furthermore, inhibition of autophagy significantly increased the production of NF-κB-dependent TNFα/IL-1β in S. aureus-infected macrophages. Collectively, these findings demonstrated, for the first time, that the PI3K/Akt-Beclin1 signaling pathway positively regulates phagocytosis and negatively mediates NF-κB-dependent inflammation in S. aureus-infected macrophages.

IL-17A modulates osteoclast precursors' apoptosis through autophagy-TRAF3 signaling during osteoclastogenesis

Osteoclasts play an important role in bone remodeling. The inflammatory cytokine IL-17A could modulate the RANKL-induced osteoclastogenesis by regulating the autophagic activity. It is well accepted that protective autophagy has an anti-apoptotic effect. It is necessary to elucidate whether IL-17A can influence the apoptosis of osteoclast precursors (OCPs) through autophagy responses during osteoclastogenesis. The results showed that apoptosis of RAW264.7-derived OCPs was promoted by high levels of IL-17A, but the opposite anti-apoptotic function was shown by low levels of IL-17A. Furthermore, the enhanced apoptosis by high levels of IL-17A was reversed by overexpression of autophagy protein Beclin1; conversely, the inhibited apoptosis by low levels of IL-17A was restored by knockdown of Beclin1. It was also found that Beclin1 suppression with Beclin1 inhibitor (spautin1) could block the reduced apoptosis by low levels of IL-17A, which was recovered by TRAF3 knockdown. Moreover, the enhanced apoptosis by high levels of IL-17A decreased following the downregulation of TRAF3. Importantly, overexpression of caspase3 further attenuated osteoclastogenesis treated by high levels of IL-17A, without significantly affecting osteoclastogenesis stimulated by low levels of IL-17A. In conclusion, IL-17A modulates apoptosis of OCPs through Beclin1-autophagy-TRAF3 signaling pathway, thereby influencing osteoclastogenesis. Therefore, our study sheds lights on the improvement of clinical strategies of dental implantation or orthodontic treatment by revealing the novel targets in the bone remodeling.

Autophagy induced by ionizing radiation promotes cell death over survival in human colorectal cancer cells

Autophagy is commonly described as a cell survival mechanism and has been implicated in chemo- and radioresistance of cancer cells. Whether ionizing radiation induced autophagy triggers tumor cell survival or cell death still remains unclear. In this study the autophagy related proteins Beclin1 and ATG7 were tested as potential targets to sensitize colorectal carcinoma cells to ionizing radiation under normoxic, hypoxic and starvation conditions. Colony formation, apoptosis and cell cycle analysis revealed that knockdown of Beclin1 or ATG7 does not enhance radiosensitivity in HCT-116 cells. Furthermore, ATG7 knockdown led to an increased survival fraction under oxygen and glutamine starvation, indicating that ionizing radiation indeed induces autophagy which, however, leads to cell death finally. These results highlight that inhibition of autophagic pathways does not generally increase therapy success but may also lead to an unfavorable outcome especially under amino acid and oxygen restriction.

Neratinib augments the lethality of [regorafenib + sildenafil

Regorafenib is approved for the treatment of colorectal cancer and hepatocellular carcinoma. In the trial NCT02466802, we have discovered that regorafenib can be safely combined with the phosphodiesterase 5 inhibitor sildenafil in advanced solid tumor patients. The present studies determined whether the approved ERBB1/2/4 and RAS downregulating drug neratinib, could enhance the lethality of [regorafenib + sildenafil]. Neratinib enhanced [regorafenib + sildenafil] lethality in a greater than additive fashion in colon cancer cells. The drug combination reduced the expression of mutant K-RAS and of multiple histone deacetylase (HDAC) proteins that required autophagosome formation. It caused green fluorescent protein or red fluorescent protein-tagged forms of K-RAS V12 to localize into large intracellular vesicles. Compared with [regorafenib + sildenafil], the three-drug combination caused greater and more prolonged activation of the ATM-AMPK-ULK-1 pathway and caused a greater suppression and prolonged inactivation of mammalian target of rapamycin, AKT, and p70 S6K. Approximately 70% of enhanced lethality caused by neratinib required ataxia-telangiectasia-mutated (ATM)-AMP-dependent protein kinase (AMPK) signaling whereas knockdown of Beclin1, ATG5, FADD, and CD95 completely prevented the elevated killing effect. Exposure of cells to [regorafenib + sildenafil] reduced the expression of the checkpoint immunotherapy biomarkers programmed death-ligand 1, ornithine decarboxylase, and indoleamine 2,3-dioxygenase-1 and increased the expression of major histocompatibility complex A (MHCA), which also required autophagosome formation. Knockdown of specific HDAC proteins recapitulated the effects observed using chemical agents. In vivo, using mouse cancer models, neratinib significantly enhanced the antitumor efficacy of [regorafenib + sildenafil]. Our data support performing a new three drug Phase I trial combining regorafenib, sildenafil, and neratinib.

BMP4 promotes hepatocellular carcinoma proliferation by autophagy activation through JNK1-mediated Bcl-2 phosphorylation

BackgroundAutophagy is a conserved catabolic process with complicated roles in tumor development. Bone morphogenetic protein 4 (BMP4), a member of the transforming growth factor (TGF-β) family of regulatory proteins, plays a crucial role in human malignancies. However, whether BMP4 contributes to the regulation of autophagy in hepatocellular carcinoma (HCC) progression remains elusive.MethodsFunctional analysis of BMP4 on HCC proliferation and autophagy was performed both in vitro and in vivo in HepG2 and HCCLM3 cells. Autophagic activity was estimated by Western blot for autophagic marker proteins and by transmission electron microscopy (TEM). Transfection of mRFP-GFP-LC3 adenovirus was applied to observe autophagic flux and high content screening was used for quantification. The signaling pathway of BMP4-regulated HCC proliferation and autophagy was investigated by Western blot.ResultsBMP4 treatment promoted HCC cells proliferation and induced autophagy. The in vivo xenograft model supported that BMP4 overexpression promoted the growth of HCC cells and autophagy induction while BMP4 knockdown exerted the opposite effect. 3-MA pre-treatment or knockdown of Beclin-1 (BECN1) blocked HCC autophagy by decreasing the expression of LC3-II and subsequently attenuated BMP4-induced autophagy and cells proliferation enhanced by BMP4 in vitro and in vivo. Mechanistic study revealed that the induction of autophagy by BMP4 was mediated through activating the JNK1/Bcl2 pathway. Furthermore, the JNK1 inhibitor and knockdown of JNK1 could attenuate autophagy induced by BMP4 and eliminated BMP4-promoted HCC cells growth.ConclusionsBMP4 promoted HCC proliferation by autophagy activation through JNK1/Bcl-2 signaling.

Knockdown of Beclin-1 impairs epithelial-mesenchymal transition of colon cancer cells.

Activation of autophagy significantly affects cancer cell behaviors, such as proliferation, differentiation, and invasiveness. Epithelial-to-mesenchymal transition (EMT) as an initial step of malignant transformation of cancer cells was linked to the activation of autophagy, but the detailed molecular mechanisms are still unknown. The present study investigates the effects of Beclin-1, a key molecule involved in activation of autophagy, on EMT of colon cancer cells. The normal colon epithelia cell line of CCD-18Co and six colon cancer cell lines with different expression levels of Beclin-1 were used in this study. The activation of autophagy and EMT markers of cancer cells were monitored by Western blotting and quantitative real-time PCR assay in the presence or absence of rapamycin (autophagy activator) and 3-MA (autophagy inhibitor). The expression of Beclin-1 in selected cell lines was modulated using small interfering RNA, and consequentially EMT markers, and cancer cell behaviors including migration and invasion, were also explored. Activation or inhibition of autophagy in colon cancer cells had positive or negative impacts on the expression of EMT markers and malignant behaviors such as cell migration and invasion. Knockdown of beclin-1 by siRNA apparently inhibited the activation of autophagy induced by rapamycin, consequentially resulted in suppression of EMT and attenuation of invasiveness of colon cancer cells. The results in this study demonstrated an association between activation of autophagy and EMT in colon cancer cells. The results showed suppression of Beclin-1 expression significantly reduced EMT and invasive behaviors in colon cancer cells.

MEG3 is involved in the development of glaucoma through promoting the autophagy of retinal ganglion cells.

In this study, we aimed at investigating whether MEG3 may be involved in the pathogenesis of glaucoma by regulating the autophagy of retinal ganglion cells (RGCs). We used qRT-PCR to detect the expression of MEG3 in RGC-5s cell line under high hydrostatic pressure. RGC-5s were transfected with a lentiviral vector to achieve MEG3 overexpression or knockdown. The influence of overexpression or inhibition of MEG3 on cell proliferation and apoptosis was observed using CCK-8 test and flow cytometry. After overexpression of MEG3 and/or knockdown of MEG3 or Beclin-1, detection of the expressions of autophagy-related and apoptosis-related proteins was performed using Western blot. MEG3 expression level increased in RGC-5 cells under high hydrostatic pressure, while exogenously decreased MEG3 expression can reverse the impact of the high pressure on RGC-5 cells. Additionally, overexpression of MEG3 can improve Atg3 expression, promote cell apoptosis, inhibit cell proliferation, and enhance autophagy levels. Meanwhile, knockdown of Beclin-1 up-regulated Bcl-2 level. Upregulation of MEG3 is involved in the pathogenesis of glaucoma through promoting apoptosis of retinal ganglion cells, the mechanism of which may be related to the enhanced autophagy levels.

In vitro and in vivo effects of MK2206 and chloroquine combination therapy on endometriosis: autophagy may be required for regrowth of endometriosis.

A high recurrence rate after medical treatment is a major clinical problem for patients with endometriosis. Here, we have evaluated the in vitro effects of combined treatment with MK2206 (an AKT inhibitor)+chloroquine on cell growth and regrowth of endometriotic stromal cells and the in vivo effects on endometriotic implants in a mouse xenograft model of endometriosis. We evaluated the effects of autophagy inhibition by knockdown of the ATG13, Beclin-1 and ATG12 genes and pharmacological agents (chloroquine, bafilomycin A1 or 3-methyalanine) individually and in combination with MK2206 on cell growth and/or cell regrowth of endometriotic stromal cells in vitro. Furthermore, we evaluated treatment with MK2206+chloroquine on endometriotic implants in a mouse xenograft model of endometriosis. Combined treatment with MK2206 and chloroquine markedly reduced cell growth and regrowth after discontinuation of treatment in endometriotic stromal cells compared with cells treated with either drug alone. Autophagy inhibition by ATG13, Beclin-1 or ATG12 gene knockdown only affected regrowth of endometriotic stromal cells, but not endometrial stromal cells from the same patients, after a 72h discontinuation of the combined treatment. Furthermore, combined treatment reduced the size of endometriotic implants, whereas no effects on endometriotic implants treated with either drug alone were observed in a mouse xenograft model of endometriosis. The present findings suggest that a novel strategy for treatment of endometriosis may involve decreasing the number of endometriotic cells that can survive treatment and then preventing regrowth by autophagy inhibition.