Ivermectin induces PAK1-mediated cytostatic autophagy in breast cancer

Ivermectin is a broad-spectrum antiparasitic drug that has recently been demonstrated to exhibit potent anticancer activity against colon cancer, ovarian cancer, melanoma and leukemia. However, the molecular mechanism underlying this anticancer effect remains poorly understood. We recently found that ivermectin markedly inhibits the growth of breast cancer cells by stimulating cytostatic macroautophagy/autophagy in vitro and in vivo. Ivermectin inhibits the AKT-MTOR signaling pathway by promoting ubiquitination-mediated degradation of PAK1 (p21 [RAC1] activated kinase 1), leading to increased autophagic flux. Together, our work unravels the molecular mechanism underpinning ivermectin-induced cytostatic autophagy in breast cancer, and characterizes ivermectin as a potential therapeutic option for breast cancer treatment.     

Glucose starvation induces autophagy via ULK1-mediated activation of PIKfyve in an AMPK-dependent manner

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Silencing of EEF2K (eukaryotic elongation factor-2 kinase) reveals AMPK-ULK1-dependent autophagy in colon cancer cells

EEF2K (eukaryotic elongation factor-2 kinase), also known as Ca²⁺/calmodulin-dependent protein kinase III, functions in downregulating peptide chain elongation through inactivation of EEF2 (eukaryotic translation elongation factor 2). Currently, there is a limited amount of information on the promotion of autophagic survival by EEF2K in breast and glioblastoma cell lines. However, the precise role of EEF2K in carcinogenesis as well as the underlying mechanism involved is still poorly understood. In this study, contrary to the reported autophagy-promoting activity of EEF2K in certain cancer cells, EEF2K is shown to negatively regulate autophagy in human colon cancer cells as indicated by the increase of LC3-II levels, the accumulation of LC3 dots per cell, and the promotion of autophagic flux in EEF2K knockdown cells. EEF2K negatively regulates cell viability, clonogenicity, cell proliferation, and cell size in colon cancer cells. Autophagy induced by EEF2K silencing promotes cell survival and does not potentiate the anticancer efficacy of the AKT inhibitor MK-2206. In addition, autophagy induced by silencing of EEF2K is attributed to induction of protein synthesis and activation of the AMPK-ULK1 pathway, independent of the suppression of MTOR activity and ROS generation. Knockdown of AMPK or ULK1 significantly abrogates EEF2K silencing-induced increase of LC3-II levels, accumulation of LC3 dots per cell as well as cell proliferation in colon cancer cells. In conclusion, silencing of EEF2K promotes autophagic survival via activation of the AMPK-ULK1 pathway in colon cancer cells. This finding suggests that upregulation of EEF2K activity may constitute a novel approach for the treatment of human colon cancer.     

PTBP1 promotes the growth of breast cancer cells through the PTEN/Akt pathway and autophagy

Invasion and migration is the hallmark of malignant tumors as well as the major cause for breast cancer death. The polypyrimidine tract binding, PTB, protein serves as an important model for understanding how RNA binding proteins affect proliferation and invasion and how changes in the expression of these proteins can control complex programs of tumorigenesis. We have investigated some roles of polypyrimidine tract binding protein 1 (PTBP1) in human breast cancer. We found that PTBP1 was upregulated in breast cancer tissues compared with normal tissues and the same result was confirmed in breast cancer cell lines. Knockdown of PTBP1 substantially inhibited tumor cell growth, migration, and invasion. These results suggest that PTBP1 is associated with breast tumorigenesis and appears to be required for tumor cell growth and maintenance of metastasis. We further analyzed the relationship between PTBP1 and clinicopathological parameters and found that PTBP1 was correlated with her‐2 expression, lymph node metastasis, and pathological stage. This will be a novel target for her‐2(⁺) breast cancer. PTBP1 exerts these effects, in part, by regulating the phosphatase and tensin homolog‐phosphatidylinositol‐4,5‐bisphosphate 3‐kinase/protein kinase B (PTEN‐PI3K/Akt) pathway and autophagy, and consequently alters cell growth and contributes to the invasion and metastasis.     

Emerging role of autophagy in mediating widespread actions of ADIPOQ/adiponectin

Autophagy can dictate changes in cell metabolism via numerous mechanisms. ADIPOQ/adiponectin has been extensively characterized to have beneficial metabolic effects, both via INS/insulin-sensitizing and INS-independent actions. Our recent work examined the regulation of skeletal muscle autophagy by ADIPOQ and the functional significance. We showed that ADIPOQ directly stimulates autophagic flux in cultured skeletal muscle cells via an AMPK-dependent signaling pathway leading to phosphorylation of ULK1 (Ser555). Pharmacological inhibition of autophagy or overexpressing an inactive mutant of ATG5 to create an autophagy-deficient cell model reduces INS sensitivity. A high-fat diet (HFD) does not induce skeletal muscle autophagy in Adipoq knockout (Ad-KO) mice, whereas it does in wild-type (WT) mice, although ADIPOQ replenishment in Ad-KO mice stimulates autophagy. Changes in skeletal muscle autophagy correlate well with peripheral INS sensitivity and glucose metabolism. Thus, ADIPOQ stimulates autophagic flux in skeletal muscle, which likely represents one important mechanism mediating multiple favorable metabolic effects.     

Ebastine exerts antitumor activity and induces autophagy by activating AMPK/ULK1 signaling in an IPMK-dependent manner in osteosarcoma

Numerous studies have confirmed that in addition to interfering with the tumor inflammatory environment, anti-inflammatory agents can directly increase apoptosis and sensitivity to conventional therapies and decrease invasion and metastasis, making them useful candidates for cancer therapy. Here, we first used high-throughput screening and had screened one compound candidate, ebastine (a H1-histamine receptor antagonist), for osteosarcoma therapy. Cell viability assays, colony formation assays, wound healing assays, and Transwell assays demonstrated that ebastine elicited antitumor effects in osteosarcoma cells. In addition, ebastine treatment exerted obvious effects on cell cycle arrest, metastasis inhibition, apoptosis and autophagy induction both in vitro and in vivo. Mechanistically, we observed that ebastine treatment triggered proapoptotic autophagy by activating AMPK/ULK1 signaling in osteosarcoma cells. Treatment with the AMPK inhibitor dorsomorphin reversed ebastine-induced apoptosis and autophagy. More importantly, we found that IPMK interacted with AMPK and functioned as a positive regulator of AMPK protein in osteosarcoma cells. A rescue study showed that the induction of autophagy and activation of the AMPK/ULK1 signaling pathway by ebastine treatment were reversed by IPMK knockdown, indicating that the activity of ebastine was IPMK dependent. We provide experimental evidence demonstrating that ebastine has antitumor activity in osteosarcoma and promotes autophagy by activating the AMPK/ULK1 signaling pathway, which is IPMK dependent. Our results provide insight into the clinical application potential of ebastine, which may represent a new potential therapeutic candidate for the treatment of osteosarcoma.     

Retracted: 9za plays cytotoxic and proapoptotic roles and induces cytoprotective autophagy through the PDK1/Akt/mTOR axis in non-small-cell lung cancer

Non-small-cell lung cancer (NSCLC) is an aggressive subtype of pulmonary carcinomas with high mortality. However, chemotherapy drug resistance and high recurrence rates hinder the curative effect of platinum-based first-line chemotherapy, which makes it urgent to develop new antitumor drugs for NSCLC. 9za, a new candidate drug synthesized by our research group, has been verified with potent antilung cancer activity in preliminary experiments. However, the underlying molecular mechanism of 9za remains largely vague. This work revealed that 9za could play important cytotoxic and proapoptotic roles in NSCLC cells. Moreover, 9za could induce autophagy and promote autophagy flux. Interestingly, the cytotoxic and proapoptotic roles were significantly dependent on 9za-induced cytoprotective autophagy. That is, the coadministration of 9za with an autophagy inhibitor such as chloroquine or 3-methyladenine exhibited increased cytotoxic and proapoptotic effects compared with 9za treatment alone. In addition, 9za exposure suppressed the phosphorylation of phosphoinositide-dependent protein kinase 1 (PDK1), protein kinase B (Akt), mammalian targets of rapamycin (mTOR), p70 S6 kinase, and 4E binding protein 1 by a dose-dependent way, manifesting that the Akt/mTOR axis was implicated in 9za-induced autophagy. In addition, the overexpression of PDK1 resulted in increased phosphorylation of PDK1 and Akt and blocking of 9za-mediated autophagy. These data showed that the PDK1/Akt/mTOR pathway was involved in 9za-induced autophagy. Hence, this work provides a theoretical basis for exploiting 9za as a new antilung cancer candidate drug and hints that the combination of 9za with an autophagy inhibitor is a feasible alternative approach for the therapy of NSCLC.     

MTA1 is a novel regulator of autophagy that induces tamoxifen resistance in breast cancer cells

Tamoxifen is commonly used to treat patients with ESR/ER-positive breast cancer, but its therapeutic benefit is limited by the development of resistance. Recently, alterations in macroautophagy/autophagy function were demonstrated to be a potential mechanism for tamoxifen resistance. Although MTA1 (metastasis-associated 1) has been implicated in breast tumorigenesis and metastasis, its role in endocrine resistance has not been studied. Here, we report that the level of MTA1 expression was upregulated in the tamoxifen resistant breast cancer cell lines MCF7/TAMR and T47D/TR, and knockdown of MTA1 sensitized the cells to 4-hydroxytamoxifen (4OHT). Moreover, knockdown of MTA1 significantly decreased the enhanced autophagy flux in the tamoxifen resistant cell lines. To confirm the role of MTA1 in the development of tamoxifen resistance, we established a cell line, MCF7/MTA1, which stably expressed MTA1. Compared with parental MCF7, MCF7/MTA1 cells were more resistant to 4OHT-induced growth inhibition in vitro and in vivo, and showed increased autophagy flux and higher numbers of autophagosomes. Knockdown of ATG7 or cotreatment with hydroxychloroquine, an autophagy inhibitor, restored sensitivity to 4OHT in both the MCF7/MTA1 and tamoxifen resistant cells. In addition, AMP-activated protein kinase (AMPK) was activated, probably because of an increased AMP:ATP ratio and decreased expression of mitochondrial electron transport complex components. Finally, publicly available breast cancer patient datasets indicate that MTA1 levels correlate with poor prognosis and development of recurrence in patients with breast cancer treated with tamoxifen. Overall, our findings demonstrated that MTA1 induces AMPK activation and subsequent autophagy that could contribute to tamoxifen resistance in breast cancer.     

Loss of STK11 expression is an early event in prostate carcinogenesis and predicts therapeutic response to targeted therapy against MAPK/p38

Prostate cancer (PCa) is the second leading cause of cancer-related death in men; however, the molecular mechanisms leading to its development and progression are not yet fully elucidated. Of note, it has been recently shown that conditional stk11 knockout mice develop atypical hyperplasia and prostate intraepithelial neoplasia (PIN). We recently reported an inverse correlation between the activity of the STK11/AMPK pathway and the MAPK/p38 cascade in HIF1A-dependent malignancies. Furthermore, MAPK/p38 overactivation was detected in benign prostate hyperplasia, PIN and PCa in mice and humans. Here we report that STK11 expression is significantly decreased in PCa compared to normal tissues. Moreover, STK11 protein levels decreased throughout prostate carcinogenesis. To gain insight into the role of STK11-MAPK/p38 activity balance in PCa, we treated PCa cell lines and primary biopsies with a well-established MAPK14-MAPK11 inhibitor (SB202190), which has been extensively used in vitro and in vivo. Our results indicate that inhibition of MAPK/p38 significantly affects PCa cell survival in an STK11-dependent manner. Indeed, we found that pharmacologic inactivation of MAPK/p38 does not affect viability of STK11-proficient PCa cells due to the triggering of the AMPK-dependent autophagic pathway, while it induces apoptosis in STK11-deficient cells irrespective of androgen receptor (AR) status. Of note, AMPK inactivation or autophagy inhibition in STK11-proficient cells sensitize SB202190-treated PCa cells to apoptosis. On the other end, reconstitution of functional STK11 in STK11-deficient PCa cells abrogates apoptosis. Collectively, our data show that STK11 is a key factor involved in the early phases of prostate carcinogenesis, and suggest that it might be used as a predictive marker of therapeutic response to MAPK/p38 inhibitors in PCa patients.     

AMPK-mTOR-ULK1 axis activation-dependent autophagy promotes hydroxycamptothecin-induced apoptosis in human bladder cancer cells

10-hydroxycamptothecin (HCPT), a natural plant extract, exerts anticancer capacity. HCPT has been reported to induce apoptosis and autophagy in human cancer cells. The interaction between autophagy and apoptosis induced by HCPT and the molecular mechanism in bladder cancer cells were investigated in this study. Our results confirmed that HCPT suppressed cell viability and migration and caused cell-cycle arrest in T24 and 5637. Then, we used Z-VAD(OMe)-FMK to clarify that apoptosis induced by HCPT was mediated by caspase. Moreover, HCPT boosted autophagy through activating the AMPK/mTOR/ULK1 pathway. Blocking autophagy by 3-methyladenine, the adenosine monophosphate-activated protein kinase (AMPK) inhibitor dorsomorphin and siATG7 reversed HCPT-induced cytotoxicity. Conversely, rapamycin and the AMPK activator AICAR enhanced growth inhibition and cell apoptosis, suggesting that autophagy played a proapoptosis role. Taken together, our findings showed that HCPT-induced autophagy mediated by the AMPK pathway in T24 and 5637 cell lines, which reinforced the apoptosis, indicating that HCPT together with autophagy activator would be a novel strategy for clinical treatment in bladder cancer.     

A small-molecule activator induces ULK1-modulating autophagy-associated cell death in triple negative breast cancer

ULK1 (unc-51 like autophagy activating kinase 1) is well known to be required to initiate the macroautophagy/autophagy process, and thus activation of ULK1-modulating autophagy/autophagy-associated cell death (ACD) may be a possible therapeutic strategy in triple negative breast cancer (TNBC). Here, our integrated The Cancer Genome Atlas (TCGA) data set, tissue microarray-based analyses and multiple biologic evaluations together demonstrate a new small-molecule activator of ULK1 for better understanding of how ULK1, the mammalian homolog of yeast Atg1, as a potential drug target can regulate ACD by the ULK complex (ULK1-ATG13-RB1CC1/FIP200-ATG101), as well as other possible ULK1 interactors, including ATF3, RAD21 and CASP3/caspase3 in TNBC. Moreover, such new inspiring findings may help us discover that this activator of ULK1 (LYN-1604) with its anti-tumor activity and ACD-modulating mechanisms can be further exploited as a small-molecule candidate drug for future TNBC therapy.     

WIPI β-propellers function as scaffolds for STK11/LKB1-AMPK and AMPK-related kinase signaling in autophagy

The article discusses new findings on the role of the 4 human WIPI proteins at the onset of macroautophagy/autophagy. New insights revealing a circuit scaffold function of WIPI β-propellers that interconnect autophagy signaling control with appropriate autophagosome formation are summarized.     

Rapamycin enhances growth inhibition on urothelial carcinoma cells through LKB1 deficiency-mediated mitochondrial dysregulation

Rapamycin, a mammalian target of rapamycin (mTOR) inhibitor, has significant potential for application in the treatment of urothelial carcinoma (URCa) of the bladder. Previous studies have shown that regulation of the AMP-activated serine/threonine protein kinase (AMPK)–mTOR signaling pathway enhances apoptosis by inducing autophagy or mitophagy in bladder cancer. Alteration of liver kinase B1 (LKB1)-AMPK signaling leads to mitochondrial dysfunction and the accumulation of autophagy-related proteins as a result of mitophagy, resulting in enhanced cell sensitivity to drug treatments. Therefore, we hypothesized that LKB1 deficiency in URCa cells could lead to increased sensitivity to rapamycin by inducing mitochondrial defect-mediated mitophagy. To test this, we established stable LKBI-knockdown URCa cells and analyzed the effects of rapamycin on their growth. Rapamycin enhanced growth inhibition and apoptosis in stable LKB1-knockdown URCa cells and in a xenograft mouse model. In spite of the stable downregulation of LKB1 expression, rapamycin induced AMPK activation in URCa cells, causing loss of the mitochondrial membrane potential, ATP depletion, and ROS accumulation, indicating an alteration of mitochondrial biogenesis. Our findings suggest that the absence of LKB1 can be targeted to induce dysregulated mitochondrial biogenesis by rapamycin treatment in the design of novel therapeutic strategies for bladder cancer.     

Acetylshikonin induces autophagy‐dependent apoptosis through the key LKB1‐AMPK and PI3K/Akt‐regulated mTOR signalling pathways in HL‐60 cells

Acetylshikonin (ASK) is a natural naphthoquinone derivative of traditional Chinese medicine Lithospermum erythrorhyzon. It has been reported that ASK has bactericidal, anti‐inflammatory and antitumour effects. However, whether ASK induces apoptosis and autophagy in acute myeloid leukaemia (AML) cells and the underlying mechanism are still unclear. Here, we explored the roles of apoptosis and autophagy in ASK‐induced cell death and the potential molecular mechanisms in human AML HL‐60 cells. The results demonstrated that ASK remarkably inhibited the cell proliferation, viability and induced apoptosis in HL‐60 cells through the mitochondrial pathway, and ASK promoted cell cycle arrest in the S‐phase. In addition, the increased formation of autophagosomes, the turnover from light chain 3B (LC3B) I to LC3B II and decrease of P62 suggested the induction of autophagy by ASK. Furthermore, ASK significantly decreased PI3K, phospho‐Akt and p‐p70S6K expression, while enhanced phospho‐AMP‐activated protein kinase (AMPK) and phospho‐liver kinase B1(LKB1) expression. The suppression of ASK‐induced the conversion from LC3B I to LC3B II caused by the application of inhibitors of AMPK (compound C) demonstrated that ASK‐induced autophagy depends on the LKB1/AMPK pathway. These data suggested that the autophagy induced by ASK were dependent on the activation of LKB1/AMPK signalling and suppression of PI3K/Akt/mTOR pathways. The cleavage of the apoptosis‐related markers caspase‐3 and caspase‐9 and the activity of caspase‐3 induced by ASK were markedly reduced by inhibitor of AMPK (compound C), an autophagy inhibitor 3‐methyladenine (3‐MA) and another autophagy inhibitor chloroquine (CQ). Taken together, our data reveal that ASK‐induced HL‐60 cell apoptosis is dependent on the activation of autophagy via the LKB1/AMPK and PI3K/Akt‐regulated mTOR signalling pathways.     

Targeting AMPK-ULK1-mediated autophagy for combating BET inhibitor resistance in acute myeloid leukemia stem cells

Bromodomain and extraterminal domain (BET) inhibitors are promising epigenetic agents for the treatment of various subsets of acute myeloid leukemia (AML). However, the resistance of leukemia stem cells (LSCs) to BET inhibitors remains a major challenge. In this study, we evaluated the mechanisms underlying LSC resistance to the BET inhibitor JQ1. We evaluated the levels of apoptosis and macroautophagy/autophagy induced by JQ1 in LSC-like leukemia cell lines and primary CD34⁺ CD38^? leukemic blasts obtained from AML cases with normal karyotype without recurrent mutations. JQ1 effectively induced apoptosis in a concentration-dependent manner in JQ1-sensitive AML cells. However, in JQ1-resistant AML LSCs, JQ1 induced little apoptosis and led to upregulation of BECN1/Beclin 1, increased LC3 lipidation, formation of autophagosomes, and downregulation of SQSTM1/p62. Inhibition of autophagy by pharmacological inhibitors or knockdown of BECN1 using specific siRNA enhanced JQ1-induced apoptosis in resistant cells, indicating that prosurvival autophagy occurred in these cells. Independent of MTOR signaling, activation of the AMPK (p-Thr172)-ULK1 (p-Ser555) pathway was found to be associated with JQ1-induced autophagy in resistant cells. AMPK inhibition using the pharmacological inhibitor compound C or by knockdown of PRKAA/AMPKα suppressed autophagy and promoted JQ1-induced apoptosis in AML LSCs. These findings revealed that prosurvival autophagy was one of the mechanisms involved in the resistance of AML LSCs to JQ1. Targeting the AMPK-ULK1 pathway or inhibition of autophagy could be an effective therapeutic strategy for combating resistance to BET inhibitors in AML and other types of cancer.     

Haemophilus parasuis infection in 3D4/21 cells induces autophagy through the AMPK pathway

Haemophilus parasuis (H. parasuis) is a common commensal in the upper respiratory tract of pigs, but causes Glässer's disease in stress conditions. To date, many studies focused on the immune evasion and virulence of H. parasuis; very few have focused on the role autophagy played in H. parasuis infection, particularly in porcine alveolar macrophages (PAMs). In this study, a PAM cell line, 3D4/21 cells were used to study the role of autophagy in H. parasuis infection. 3D4/21 cells tandemly expressing GFP, mCherry, and LC3 were infected with H. parasuis serovar 5 (Hps5). Western blot analysis and confocal and transmission electron microscopy showed that H. parasuis infection effectively induces autophagy. Using Hps strains of varying virulence (Hps4, Hps5, and Hps7) and UV‐inactivated Hps5, we demonstrated that autophagy is associated with the internalisation of living virulent strains into cells. In 3D4/21 cells pretreated with rapamycin and 3‐MA then infected by Hps4, Hps5, and Hps7, we demonstrated that autophagy affects invasion of H. parasuis in cells. AMPK signal results showed that Hps5 infection can upregulate the phosphorylation level of AMPK, which is consistent with the autophagy development. 3D4/21 cells pretreated with AICAR or Compound C then infected by Hps5 revealed that the autophagy induced by Hps5 infection is associated with the AMPK pathway. Our study contributes to the theoretical basis for the study of H. parasuis pathogenesis and development of novel drugs target for prevention Glässer's disease.     

Critical role of CAV1/caveolin-1 in cell stress responses in human breast cancer cells via modulation of lysosomal function and autophagy

CAV1 (caveolin 1, caveolae protein, 22kDa) is well known as a principal scaffolding protein of caveolae, a specialized plasma membrane structure. Relatively, the caveolae-independent function of CAV1 is less studied. Autophagy is a process known to involve various membrane structures, including autophagosomes, lysosomes, and autolysosomes for degradation of intracellular proteins and organelles. Currently, the function of CAV1 in autophagy remains largely elusive. In this study, we demonstrate for the first time that CAV1 deficiency promotes both basal and inducible autophagy. Interestingly, the promoting effect was found mainly in the late stage of autophagy via enhancing lysosomal function and autophagosome-lysosome fusion. Notably, the regulatory function of CAV1 in lysosome and autophagy was found to be caveolae-independent, and acts through lipid rafts. Furthermore, the elevated autophagy level induced by CAV1 deficiency serves as a cell survival mechanism under starvation. Importantly, downregulation of CAV1 and enhanced autophagy level were observed in human breast cancer cells and tissues. Taken together, our data reveal a novel function of CAV1 and lipid rafts in breast cancer development via modulation of lysosomal function and autophagy.