Treatment-induced Autophagy Research Articles

Abstract 4322: Inhibition of ULK and KRASG12C control tumor growth in preclinical models of lung cancer

Abstract Approximately 30% of lung cancers are driven by mutationally activated KRAS. The recent FDA approval of sotorasib, a direct pharmacological inhibitor of KRASG12C, marks a critical milestone in the treatment of this important subset of lung cancer. However, a major obstacle in treating lung cancer is resistance to current therapeutic treatments. In response to cellular stresses such as RAS pathway-targeted therapeutics, RAS-mutated cancer cells have been demonstrated to increase autophagy, an intracellular recycling pathway. However, the mechanism(s) underlying treatment-induced autophagy is not well understood. Here, we demonstrate that KRASG12C-driven lung cancer cells increase autophagy in response to treatment with sotorasib. Currently, the only therapeutics in clinical use to inhibit autophagy are lysosomal inhibitors (hydroxy)chloroquine. High concentrations of these compounds are needed to achieve modest inhibition of autophagy, suggesting that the potency of these compounds may limit clinical responses. ULK kinases are serine/threonine kinases that are necessary for the initiation of autophagy. DCC-3116 is a potent inhibitor of the ULK1/2 protein kinases, master regulators of autophagy. Treatment with sotorasib and DCC-3116 led to superior anti-tumor effects in preclinical models of lung cancer. Additionally, about 30% of lung cancer patients with KRAS mutations also have deletions or inactivating mutations in LKB1, a protein involved in the regulation of nutrient sensing and autophagy. In other KRAS-driven cancers, the LKB1-AMPK-ULK1 signaling axis is a proposed mechanism as to how autophagic flux increases following KRAS pathway inhibition. However, our preliminary data demonstrates that LKB1 is dispensable for sotorasib-induced autophagy, suggesting the mechanism(s) of autophagy induction in KRASG12C-driven lung cancer may be different from those detected in other KRAS mutated cancers. Consequently, we have generated genetically engineered mouse models (GEMMs) of KRASG12C-driven lung cancer in which LKB1 is silenced to further investigate the role of LKB1 in the autophagy response of KRASG12C-driven lung cancers to pathway-targeted blockade of oncogenic KRAS signaling. We have treated KRASG12C-driven GEMMs with sotorasib and/or DCC-3116 to test the sensitivity of lung tumors to these treatment options. Citation Format: Phaedra Ghazi, Kayla O'Toole, Sanjana Boggaram, Michael Scherzer, Madhumita Bogdan, Bryan Smith, Dan Flynn, Conan Kinsey, Martin McMahon. Inhibition of ULK and KRASG12C control tumor growth in preclinical models of lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4322.

The Newly Proposed Mechanism of Cardiomyocyte Protection of Carvedilol- Anti-Apoptosis Pattern of Carvedilol in Anoxia by Inducing Autophagy Partly through the AMPK/mTOR Pathway

Purpose: To investigate the underlying mechanism of cardiomyocyte protection of carvedilol based on autophagy and apoptosis. Methods: Neonatal rat ventricular myocytes (NRVMs) were exposed to various concentrations of carvedilol before anoxia, and pretreated with 3-MA or compound C for inhibiting autophagy or p-AMPK expression. CCK-8 colorimeter and flow cytometry were used to determine the cell viability and apoptotic rates. The variation of mRNA and protein was measured by RT-PCR and Western blot. The presence of autophagosomes was observed by electron microscopy. Results: First, we found that carvedilol increased autophagic marker levels in a concentration-dependent manner and the number of autophagosomes in NRVMs. Moreover, carvedilol substantially enhanced the viability and noticeably reduced the CK, MDA and LDH levels and cell apoptosis rate compared with the anoxia group. In addition, carvedilol decreased the levels of caspase-3 and Bim in mRNA and protein, but such effect was blocked by the special autophagy inhibitor-3-MA, and the number of autophagosomes was significantly decreased when treated with 3-MA, indicating that carvedilol exhibited anti-apoptotic and anti-injury effects by inducing autophagy in anoxia NRVMs, but these effects can be abolished by adding 3-MA to suppress autophagy. Finally, the carvedilol treatment-induced autophagy by enhancing the activation of p-AMPK and inhibiting p-mTOR. Electron microscopy presented that the number of autophagosomes was significantly decreased when treating with compound C, indicating that carvedilol induced autophagy in anoxia NRVMs partly by the AMPK-mTOR signaling pathway. Conclusions: Carvedilol has cardioprotection by inducing autophagy against apoptosis partly through the AMPK/mTOR pathway during anoxia in NRVMs.

Autophagy Inhibition with Chloroquine Increased Pro-Apoptotic Potential of New Aziridine-Hydrazide Hydrazone Derivatives against Glioblastoma Cells.

Tumor therapy escape due to undesired side effects induced by treatment, such as prosurvival autophagy or cellular senescence, is one of the key mechanisms of resistance that eventually leads to tumor dormancy and recurrence. Glioblastoma is the most frequent and practically incurable neoplasm of the central nervous system; thus, new treatment modalities have been investigated to find a solution more effective than the currently applied standards based on temozolomide. The present study examined the newly synthesized compounds of aziridine-hydrazide hydrazone derivatives to determine their antineoplastic potential against glioblastoma cells in vitro. Although the output of our investigation clearly demonstrates their proapoptotic activity, the cytotoxic effect appeared to be blocked by treatment-induced autophagy, the phenomenon also detected in the case of temozolomide action. The addition of an autophagy inhibitor, chloroquine, resulted in a significant increase in apoptosis triggered by the tested compounds, as well as temozolomide. The new aziridine-hydrazide hydrazone derivatives, which present cytotoxic potential against glioblastoma cells comparable to or even higher than that of temozolomide, show promising results and, thus, should be further investigated as antineoplastic agents. Moreover, our findings suggest that the combination of an apoptosis inducer with an autophagy inhibitor could optimize chemotherapeutic efficiency, and the addition of an autophagy inhibitor should be considered as an optional adjunctive therapy minimizing the risk of tumor escape from treatment.

Abstract PR03: Cooperative anti-tumor effects of combined inhibition of KRASG12C plus autophagy in preclinical models of KRASG12C-driven lung cancer

Abstract Approximately 25% of lung cancers are driven by mutationally-activated KRAS. A major obstacle in treating lung cancer is resistance to current therapeutic treatments. The recent FDA approval of sotorasib, a direct pharmacological inhibitor of KRASG12C, marks a critical milestone in the treatment of this important subset of lung cancer. However, the twin problems of primary or acquired resistance to direct pharmacological inhibition of KRASG12C remain major obstacles in sustaining the deepest and most durable patient responses. In response to cellular stresses such as RAS pathway-targeted therapeutics, RAS-mutated cancer cells have been demonstrated to increase autophagy, an intracellular recycling pathway. Moreover, combined inhibition of RAS pathway signaling plus autophagy had potent anti-tumor effects in preclinical models of melanoma or pancreatic cancer. However, the mechanism(s) underlying treatment-induced autophagy is not well understood. Here, we demonstrate that KRASG12C-driven lung cancer cells increase autophagic flux in response to treatment with KRASG12C inhibitors. Moreover, combined treatment of such cells with a specific, selective and potent inhibitor of the ULK1/2 protein kinases, master regulators of autophagy, led to superior anti-tumor effects in human cell line xenograft models. Previous work to understand why inhibiting autophagy sensitizes KRAS mutant cancer cells to targeted therapy has been confounded by the use of non-specific lysosomal inhibitors such as hydroxychloroquine. Using selective autophagy inhibitors, such as ULK1/2i, provides a unique opportunity to investigate the precise mechanism as to why inhibition of autophagy sensitizes cells to KRASG12C inhibition. Additionally, about 30% of lung cancer patients with KRAS mutations also have deletions or inactivating mutations in LKB1, a protein involved in regulation of nutrient sensing and autophagy. Patients with KRAS-mutated lung cancer whose tumors also lack LKB1 expression are characterized by aggressive behavior and resistance to standard treatment. In other KRAS-driven cancers, the LKB1>AMPK>ULK1 signaling axis is a proposed mechanism as to how autophagic flux increases following KRAS pathway inhibition. However, our preliminary data suggests that LKB1 is dispensable for sotorasib-induced autophagy in KRASG12C-driven lung cancer cells. To this end, KRASG12C-driven lung cancer cell lines with loss of LKB1 expression significantly increased autophagic flux after treatment with either a KRASG12C inhibitor or inhibitors of RAF>MEK>ERK signaling suggesting that the mechanism(s) of autophagy induction in KRASG12C-driven may be different to those detected in other KRAS mutated cancers. Consequently, we have generated new genetically engineered mouse models of KRASG12C-driven lung cancer in which LKB1 is silenced to further investigate the role of LKB1 in the autophagy response of KRASG12C-driven lung cancers to pathway-targeted blockade of oncogenic KRAS signaling. Citation Format: Phaedra C. Ghazi, Conan Kinsey, Madhumita Bogdan, Bryan D. Smith, Daniel L. Flynn, Martin McMahon. Cooperative anti-tumor effects of combined inhibition of KRASG12C plus autophagy in preclinical models of KRASG12C-driven lung cancer [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr PR03.

Targeting Unc51-like Autophagy Activating Kinase 1 (ULK1) Overcomes Adaptive Drug Resistance in Acute Myelogenous Leukemia.

ULK1 drives a cell-intrinsic adaptive resistance in AML and targeting ULK1-mediated autophagy can synergize with existing and emerging AML therapies to overcome drug-resistance and induce apoptosis.

Heat treatment-induced autophagy promotes breast cancer cell invasion and metastasis via TGF-β2-mediated epithelial-mesenchymal transitions.

Insufficient thermal ablation can accelerate malignant behaviors and metastases in some solid tumors, and epithelial-mesenchymal transition (EMT) and autophagy are involved in tumor metastasis. It has been found that TGF-β2 which belongs to the family of transforming growth factors often associated with cancer cell invasiveness and EMT. However, whether the interactions between autophagy and TGF-β2 induce EMT in breast cancer (BC) cells following insufficient microwave ablation (MWA) remains unclear. BC cells were treated with sublethal heat treatment to simulate insufficient MWA, and the effects of heat treatment on the BC cell phenotypes were explored. CCK-8, colony formation, flow cytometry, Transwell, and wound healing assays were performed to evaluate the influence of sublethal heat treatment on the proliferation, apoptosis, invasion, and migration of BC cells. Western blotting, real-time quantitative PCR, immunofluorescence, and transmission electron microscopy were carried out to determine the changes in markers associated with autophagy and EMT following sublethal heat treatment. Results showed that heat treatment promoted the proliferation of surviving BC cells, which was accompanied by autophagy induction. Heat treatment-induced autophagy up-regulated TGF-β2/Smad2 signaling and promoted EMT phenotype, thereby enhancing BC cells' migration and invasion abilities. An increase or decrease of TGF-β2 expression resulted in the potentiation and suppression of autophagy, as well as the enhancement and abatement of EMT. Autophagy inhibitors facilitated apoptosis and repressed proliferation of BC cells in vitro, and thwarted BC cell tumor growth and pulmonary metastasis in vivo. Heat treatment-induced autophagy promoted invasion and metastasis via TGF-β2/Smad2-mediated EMTs. Suppressing autophagy may be a suitable strategy for overcoming the progression and metastasis of residual BC cells following insufficient MWA.

The mechanisms of arsenic-induced ovotoxicity, ultrastructural alterations, and autophagic related paths: An enduring developmental study in folliculogenesis of mice

Arsenic (As) exerts a wide range of adverse effects on biological systems, including the reproductive organs in males and females. However, the mechanisms of As-induced reproductive toxicity are mostly obscure. Recently, we showed that autophagy is an essential route for As2O3-induced reprotoxicity through the hypothalamic-pituitary-gonadal-sperm (HPG-S) axis in pubertal and matured F1-male mice. However, the role of autophagy in As2O3- induced ovarian toxicity is mostly unknown. Hence, this study aimed to elucidate the role of oxidative stress, mitochondrial impairment, and autophagic processes in the ovary of As-exposed female mice. For this purpose, mature female mice were challenged with 0, low (0.2), medium (2), and high (20 ppm) As2O3 from 35-days before mating till weaning their pups, and the F1- females from weaning until maturity. Then, all the mice were sacrificed, and oxidative stress parameters, mitochondrial indices, electron microscopic evaluation of the ovaries, expression of autophagic-related genes and proteins, and autophagosome formation were assessed. It was shown that medium and high As2O3 doses were a potent inducer of oxidative stress, mitochondrial dysfunction, and autophagy in the ovary of F1-generation. A dose-dependent increment in the gene expression of PDK1, PI3K, TSC2, AMPK, ULK1, ATG13, Beclin1, ATG12, ATG5, LC3, P62, ATG3, ATG7, and p62, as well as protein expression of Beclin1, and LC3- I, II, was evident in the ovaries of the As-treated animals. Moreover, a dose-dependent decrease in the expression of mTOR and Bcl-2 genes, and mTOR protein was detected with increasing doses of As, suggesting that As treatment-induced autophagy. Along with a dose-dependent increase in the number of MDC-labeled autophagic vacuoles, transmission electron microscopy also confirmed more autophagosomes and injured mitochondria in medium and high As2O3 doses groups. As2O3 also negatively affected the mean body weight, litter size, organ coefficient, and stereological indices in female mice. Finally, in physiological conditions, arsenic trioxide (As2O3) leads to an increased level of autophagy in the oocyte when many oocytes were being lost. These findings indicated that an imbalance in the oxidant-antioxidant system, mitochondrial impairment, and the autophagic process, through inhibition of mTOR, dependent and independent pathways, and Bcl-2, as well as activation of AMPK/PI3K/Beclin1/LC3 routes, could play a pivotal role in As-induced reproductive toxicity through ovarian dysfunction in females.

Improved Survival in Hepatocellular Carcinoma Patients with Cardiac Arrhythmia by Amiodarone Treatment through Autophagy.

Hepatocellular carcinoma (HCC) is a primary malignancy of the liver. In basic studies, the regulation of autophagy has offered promising results for HCC treatment. This study aimed to address the question of whether amiodarone can improve survival rates in HCC patients associated with autophagy. Using datasets from the National Health Insurance Research Database, we enrolled patients over 18 years of age that had been diagnosed with HCC between January 1997 and December 2010. Amiodarone and non-amiodarone users were matched at a 1:1 frequency, according to all variables. Additionally, HepG2 cells treated with amiodarone were evaluated by cell viability and autophagic change. Autophagic signaling was examined by immunoblotting and tissue array immunohistochemistry. Of the 10,946 patients diagnosed with HCC, each cohort included 221 patients after 1:1 propensity score matching. The median survival was 36.70 months for the amiodarone users, and 24.48 months for the non-amiodarone users. After adjusting for age, gender, comorbidities and treatment, amiodarone users had a significantly lower risk of mortality. Amiodarone users also demonstrated an improved 3-year survival rate. Furthermore, amiodarone treatment-induced autophagy in HepG2 cells was demonstrated by autophagosome formation associated with increasing LC3B-II, P62, and Beclin-1 expression. Autophagic flux also increased following amiodarone treatment with bafilomycin A1. SiRNA of LC3B knocked down endogenous LC3B formation and restored HepG2 cell viability. This study provides epidemiologic evidence that amiodarone via autophagic degradation machinery may offer survival benefits for HCC patients with a history of arrhythmia. Further randomized, blinded, and placebo-controlled trials are warranted for patients with HCC.

Depletion of NBR1 in urothelial carcinoma cells enhances rapamycin-induced apoptosis through impaired autophagy and mitochondrial dysfunction.

Rapamycin is well-recognized in the clinical therapeutic intervention for patients with cancer by specifically targeting mammalian target of rapamycin (mTOR) kinase. Rapamycin regulates general autophagy to clear damaged cells. Previously, we identified increased expression of messenger RNA levels of NBR1 (the neighbor of BRCA1 gene; autophagy cargo receptor) in human urothelial cancer (URCa) cells, which were not exhibited in response to rapamycin treatment for cell growth inhibition. Autophagy plays an important role in cellular physiology and offers protection against chemotherapeutic agents as an adaptive response required for maintaining cellular energy. Here, we hypothesized that loss of NBR1 sensitizes human URCa cells to growth inhibition induced by rapamycin treatment, leading to interruption of protective autophagic activation. Also, the potential role of mitochondria in regulating autophagy was tested to clarify the mechanism by which rapamycin induces apoptosis in NBR1-knockdown URCa cells. NBR1-knockdown URCa cells exhibited enhanced sensitivity to rapamycin associated with the suppression of autophagosomal elongation and mitochondrial defects. Loss of NBR1 expression altered the cellular responses to rapamycin treatment, resulting in impaired ATP homeostasis and an increase in reactive oxygen species (ROS). Although rapamycin treatment-induced autophagy by adenosine monophosphate-activated protein kinase (AMPK) phosphorylation in NBR1-knockdown cells, it did not process the conjugated form of LC3B-II after activation by unc-51 like autophagy-activating kinase 1 (ULK1). NBR1-knockdown URCa cells exhibited rather profound mitochondrial dysfunctions in response to rapamycin treatment as evidenced by Δψm collapse, ATP depletion, ROS accumulation, and apoptosis activation. Therefore, our findings provide a rationale for rapamycin treatment of NBR1-knockdown human urothelial cancer through the regulation of autophagy and mitochondrial dysfunction by regulating the AMPK/mTOR signaling pathway, indicating that NBR1 can be a potential therapeutic target of human urothelial cancer.

Studies on the non-invasive anticancer remedy of the triple combination of epigallocatechin gallate, pulsed electric field, and ultrasound.

Cancer is one of the most troublesome diseases and a leading cause of death worldwide. Recently, novel treatments have been continuously developed to improve the disadvantages of conventional therapies, such as prodigious expenses, unwanted side effects, and tumor recurrence. Here, we provide the first non-invasive treatment that has combined epigallocatechin gallate (EGCG), the most abundant catechin in green tea, with a low strength pulsed electric field (PEF) and a low energy ultrasound (US). It has been observed that the cell viability of human pancreatic cancer PANC-1 was decreased approximately to 20% of the control after this combination treatment for 72 h. Besides, the combined triple treatment significantly reduced the high tolerance of HepG2 cells to the EGCG-induced cytotoxicity and similarly exhibited compelling proliferation-inhibitory effects. We also found the combined triple treatment increased the intracellular reactive oxygen species (ROS) and acidic vesicles, and the EGCG-induced inhibition of Akt phosphorylation was dramatically intensified. In this study, the apoptosis inhibitor Z-VAD-FMK and the autophagy inhibitor 3-MA were, respectively, shown to attenuate the anticancer effects of the triple treatment. This indicates that the triple treatment-induced autophagy was switched from cytoprotective to cytotoxic, and hence, cooperatively caused cell death with the apoptosis. Since the EGCG is easily accessible from the green tea and mild for a long-term treatment, and the non-invasive physical stimulations could be modified to focus on a specific location, this combined triple treatment may serve as a promising strategy for anticancer therapy.

Metabolic reprogramming ensures cancer cell survival despite oncogenic signaling blockade.

There is limited knowledge about the metabolic reprogramming induced by cancer therapies and how this contributes to therapeutic resistance. Here we show that although inhibition of PI3K-AKT-mTOR signaling markedly decreased glycolysis and restrained tumor growth, these signaling and metabolic restrictions triggered autophagy, which supplied the metabolites required for the maintenance of mitochondrial respiration and redox homeostasis. Specifically, we found that survival of cancer cells was critically dependent on phospholipase A2 (PLA2) to mobilize lysophospholipids and free fatty acids to sustain fatty acid oxidation and oxidative phosphorylation. Consistent with this, we observed significantly increased lipid droplets, with subsequent mobilization to mitochondria. These changes were abrogated in cells deficient for the essential autophagy gene ATG5 Accordingly, inhibition of PLA2 significantly decreased lipid droplets, decreased oxidative phosphorylation, and increased apoptosis. Together, these results describe how treatment-induced autophagy provides nutrients for cancer cell survival and identifies novel cotreatment strategies to override this survival advantage.

Magnetic Resonance Spectroscopy to Study Glycolytic Metabolism During Autophagy.

Cancer cells undergoing starvation- and treatment-induced autophagy were found to exhibit reduced intracellular lactate, reduced rates of steady-state lactate excretion and reduced real-time pyruvate-lactate exchange rates, indicating that glycolytic metabolism was altered in autophagic cells. In this chapter, we describe the technical details of the use of 1H-magnetic resonance spectroscopy (MRS) to measure endogenous cellular concentrations of lactate and glucose in autophagic cells and tissues, how to measure the rate of steady-state lactate excretion and glucose uptake by 1H-MRS in autophagic cells, and details of the real-time measurement of [1-13C] pyruvate to lactate exchange in autophagic cells by 13C-MRS-DNP (dynamic nuclear polarization).

Abstract 271: Influence of autophagy modulation on synergistic interactions of lapatinib and mTOR targeted agents in HER2-amplified lapatinib resistant breast cancer models in vitro and in vivo

Abstract Background: Resistance against HER2 targeted agents ultimately limits the therapeutic success in patients with HER2-positive breast cancer. It was shown that PIK3CA mutations contribute to lapatinib resistance and achieving control of a downstream PI3K/mTOR signaling is necessary for optimal effectiveness of HER2 blockade. We and others have shown that catalytic mTORC1/2 inhibitors reverse lapatinib resistance and inhibit growth of HER2-overexpressing breast cancer models in vitro and in vivo. However, activity of these targeted agents is hindered by the compensatory and adaptive mechanisms that arise to assure cell survival. One of the pro-survival responses is cytoprotective autophagy induced by lapatinib and mTORC1/2 inhibitors. Thus, we examined whether impairing autophagy could augment activity of lapatinib/mTORC1/2 inhibitors combinations in lapatinib-resistant breast cancer models. Methods: The combination of lapatinib with catalytic mTORC1/2 inhibitors KU-0063794 (KU) or AZD2014 (AZD) was evaluated in vitro and in vivo in lapatinib resistant PIK3CA mutated HER2-overexpressing/amplified MDA-MB-361, JIMT-1 and MDA-MB-453 breast cancer models in the presence of siRNA-based (Atg7, Beclin-1) and pharmacological (hydroxychloroquine (HCQ)) inhibitors of autophagy. Results: In vitro lapatinib/mTORC1/2 combinations elevated autophagy to a greater extent that either compound alone. Genetic or pharmacological inhibition of treatment-induced autophagy further decreased cell viability, suggesting that autophagy was playing a cytoprotective role in this context. In vivo, lapatinib and AZD combinations achieved effective tumor growth inhibition of 98%, 111% and 152% in MDA-MB-361, JIMT-1 and MDA-MB-453 models respectively, however addition of HCQ did not significantly enhance this therapeutic response (p>0.05). Conclusion: Negligible effects of HCQ in vivo in tumors treated with lapatinib/AZD combinations may be attributed to ineffective inhibition of autophagy-mediated survival signals that, if significantly blocked, could increase efficacy of the treatment. Utilizing carrier nanotechnology to optimize delivery of HCQ to the tumor site and molecular analysis of HCQ-engendered off target effects on survival and proliferation pathways in tumor tissue are being pursued. Citation Format: Wieslawa H. Dragowska, Sherry A. Weppler, William Wei Chu, Norman S. Chow, Jenna S. Rawji, Ashleen S. Prasad, Karen A. Gelmon, Sharon M. Gorski, Marcel B. Bally. Influence of autophagy modulation on synergistic interactions of lapatinib and mTOR targeted agents in HER2-amplified lapatinib resistant breast cancer models in vitro and in vivo. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 271.

Abstract B56: Treatment-induced autophagy increases amino acid uptake and switches glucose addiction to amino acid catabolism in cancer

Abstract Background: Autophagy is a cellular catabolic degradation response to starvation or stress and the rerouting of cellular metabolism by autophagic cancer cells, to sustain cellular homeostasis during starvation or treatment-induced stress, remains poorly understood. We investigated the hypotheses that treatment-induced autophagy could prolong cancer cell survival, supported by adapted metabolism reversibly maintaining cellular homeostasis. Methods: We induced autophagy in human prostate (PC3) and colorectal (HT29, HCT116 WT (wild-type) and HCT116 Bax-ko (Bax-knock out)) cancer cells by 6 or 24 hours of amino-acid and serum deprivation (in Hank's balanced salt solution), or by 24 hours of treatment with PI103 (a phosphatidyl inositol-3 kinase inhibitor) or dichloroacetate (DCA, a pyruvate dehydrogenase kinase inhibitor). Cellular metabolism during starvation- or treatment-induced autophagy and subsequent recovery were examined by 1H- and 13C-magnetic resonance spectroscopy metabolomic studies. 13C-labeled glucose was used to assess metabolic flux. In vitro findings were verified in two corresponding colorectal xenograft models treated with PI103 or DCA. Results and Discussion: Increased expression of LC3II by western blots and the increased level of autophagosomes visualized by electron microscopy confirmed the induction of autophagy with minimal apoptosis and necrosis in HCT116 Bax-ko cells following starvation, DCA or PI103 treatment and in HCT116 WT, PC3 and HT29 cells following DCA treatment. PI103-induced autophagy prolonged cell survival, whereas starvation-induced autophagic cells eventually died. In PI103- or DCA-induced autophagy, metabolism was re-routed by i) reduced aerobic glycolysis with unchanged glucose uptake, increased cellular glucose levels (P<0.003) and reduced lactate excretion (P<0.0001); ii) increased uptake of branched-chain amino acids and glutamine (P<0.005), with a net accumulation of many intracellular amino acids and succinate (P<0.003), a TCA cycle intermediate. These metabolic alterations can prolong cancer cell survival during stress in a well-nourished environment by providing energy from amino-acid catabolism. Metabolic changes were reversed on recovery from treatment-induced autophagy. Increased levels of glutamine (P<0.01) and TCA-cycle intermediates (P<0.04) were also observed in DCA- and PI103-treated HT29 and HCT116-Bax-ko tumor xenografts, providing potential non-invasive biomarkers for treatment-induced autophagy. This work is supported by the CR-UK and EPSRC Cancer Imaging Centre in association with the MRC and Department of Health (England) grants C1060/A10334 and C1060/A16464, NHS funding to the NIHR Biomedical Research Centre. Chang Gung Medical Foundation (Taiwan) grant CMRPG370441 and MRC-funded studentship (MRC119X). MOL is an NIHR Senior Investigator. We thank Alice Warley at the Kings College London Centre for Ultrastructural Imaging (CUI) for providing facilities for electron microscopy. Citation Format: Gigin Lin, Helen Troy, Gabriela Andrejeva, Anne-Christine LF Wong Te Fong, Dow-Mu Koh, Simon P. Robinson, Ian R. Judson, John R. Griffiths, Martin O. Leach, Yuen-Li Chung. Treatment-induced autophagy increases amino acid uptake and switches glucose addiction to amino acid catabolism in cancer. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr B56.

Dual inhibition of Type I and Type III PI3 kinases increases tumor cell apoptosis in HER2+ breast cancers.

IntroductionHuman epidermal growth factor receptor-2 (HER2) gene amplification (HER2+) drives tumor cell growth and survival in ~25 % of breast cancers. HER2 signaling activates the type I phosphoinositide 3-kinase (PI3K), upon which these tumors rely. Consequently, inhibitors of HER2 and type I PI3K block growth and increase apoptosis in HER2+ breast cancers, especially when used in combination. However, the impact of type III PI3K inhibition, particularly in combination with HER2 blockade or type I PI3K inhibition, remains less clear.MethodsWe utilized small molecule kinase inhibitors, locked nucleic acid antisense oligonucleotides (LNA-ASOs), and siRNA to assess proliferation, autophagy, apoptosis, and protein expression in cell culture models of HER2+ breast cancers.ResultsTreatment of HER2+ breast cancer cells with HER2 inhibitors or type I PI3K kinase inhibitors, alone or in combination, blocked type I PI3K signaling, reduced tumor cell growth, and induced autophagy. Knockdown of the type I PI3K, p110α, using an LNA-ASO termed EZN4150 inhibited PI3K-mediated Akt phosphorylation. However, in contrast to catalytic inhibitors of type I PI3Ks, EZN4150 did not induce autophagy, and blocked autophagy in response to inhibitors of HER2 or type I PI3Ks in a dominant fashion. Sequence analysis of EZN4150 revealed significant homology to the gene encoding the type III PI3K, Vps34, a key component for autophagy induction. EZN4150 simultaneously reduced expression of both p110α and Vps34. Combined inhibition of PI3K signaling and autophagy using individual siRNAs against p110α and Vps34 or using pharmacological type I and type III PI3K inhibitors recapitulated what was seen with EZN4150, and robustly enhanced tumor cell killing.ConclusionsThese studies highlight the important role of Vps34-mediated autophagy in limiting the anti-tumor response to inhibitors of HER2 or type I PI3K in HER2+ breast cancers. The type III PI3K Vps34 represents a potential therapeutic target to block treatment-induced autophagy and enhance tumor cell killing.Electronic supplementary materialThe online version of this article (doi:10.1186/s13058-015-0656-2) contains supplementary material, which is available to authorized users.

Activation of Autophagy Rescues Amiodarone-Induced Apoptosis of Lung Epithelial Cells and Pulmonary Toxicity in Rats

Amiodarone, bi-iodinated benzofuran derivative, is one of the most frequently prescribed and efficacious antiarrhythmic drugs. Despite its low incidence, amiodarone-induced pulmonary toxicity is of great concern and the leading cause of discontinuation. Autophagy is an essential homeostatic process that mediates continuous recycling of intracellular materials when nutrients are scarce. It either leads to a survival advantage or initiates death processes in cells under stress. In the present study, we investigated the role of autophagy in amiodarone-induced pulmonary toxicity. Amiodarone treatment-induced autophagy in H460 human lung epithelial cells and BEAS-2B normal human bronchial epithelial cells was demonstrated by increased LC3-II conversion, Atg7 upregulation, and autophagosome formation. Autophagic flux, as determined by the lysosomal inhibitor bafilomycin A1, was also increased following amiodarone treatment. To determine the role of autophagy in amiodarone toxicity, amiodarone-induced cell death was evaluated in the presence of 3-methyladenine or by knocking down the autophagy-related genes Atg7. Inhibition of autophagy decreased cellular viability and significantly increased apoptosis. Intratracheal instillation of amiodarone in rats increased the number of inflammatory cells recovered from bronchoalveolar lavage fluid, and periodic acid-Schiff-positive staining in bronchiolar epithelial cells. However, induction of autophagy by rapamycin treatment inhibited amiodarone-induced pulmonary toxicity. In conclusion, amiodarone treatment induced autophagy, which is involved in protection against cell death and pulmonary toxicity.

Combination treatment with arsenic trioxide and irradiation enhances autophagic effects in U118-MG cells through increased mitotic arrest and regulation of PI3K/Akt and ERK1/2 signaling pathways

Malignant gliomas are resistant to many kinds of treatments including chemotherapy, radiotherapy, and other adjuvant therapies. Autophagy is a novel response of cancer cells to ionizing radiation (IR) or chemotherapy, but its significance and underlying mechanism remains largely elusive. Induction of autophagy in glioma cells using irradiation and arsenic trioxide (ATO) have been reported separately. However, the combined effects of ATO and IR on the cell death processes of malignant glioma cells have not been thoroughly studied, especially in U118-MG cells. In the present study, we investigated the anticancer effect of IR combined with ATO and the underlying mechanisms on U118-MG human malignant glioma cells in vitro. We found that the enhanced cytotoxic effect of IR combined with ATO was through induction of more autophagy in U118-MG cells, which were characterized by the presence of acidic vascular organelle formation, determined by electron microscopic observation and immunoblotting of LC3. Combined treatment could induce more mitotic arrest compared to ATO or IR alone. In addition, we also found that the combined treatment-induced autophagy occurred through inhibition of PI3K/Akt and activation of ERK1/2 signaling pathways. These findings suggest a potential therapeutic strategy for malignant gliomas, which are resistant to various proapoptotic therapies.