Autophagy-mediated Cancer Cell Death Research Articles

A novel combination therapy with Cabozantinib and Honokiol effectively inhibits c-Met-Nrf2-induced renal tumor growth through increased oxidative stress

Receptor tyrosine kinase (RTK), c-Met, is overexpressed and hyper active in renal cell carcinoma (RCC). Most of the therapeutic agents mediate cancer cell death through increased oxidative stress. Induction of c-Met in renal cancer cells promotes the activation of redox-sensitive transcription factor Nrf2 and cytoprotective heme oxygenase-1 (HO-1), which can mediate therapeutic resistance against oxidative stress. c-Met/RTK inhibitor, Cabozantinib, has been approved for the treatment of advanced RCC. However, acquired drug resistance is a major hurdle in the clinical use of cabozantinib. Honokiol, a naturally occurring phenolic compound, has a great potential to downregulate c-Met-induced pathways. In this study, we found that a novel combination treatment with cabozantinib + Honokiol inhibits the growth of renal cancer cells in a synergistic manner through increased production of reactive oxygen species (ROS); and it significantly facilitates apoptosis-and autophagy-mediated cancer cell death. Activation of c-Met can induce Rubicon (a negative regulator of autophagy) and p62 (an autophagy adaptor protein), which can stabilize Nrf2. By utilizing OncoDB online database, we found a positive correlation among c-Met, Rubicon, p62 and Nrf2 in renal cancer. Interestingly, the combination treatment significantly downregulated Rubicon, p62 and Nrf2 in RCC cells. In a tumor xenograft model, this combination treatment markedly inhibited renal tumor growth in vivo; and it is associated with decreased expression of Rubicon, p62, HO-1 and vessel density in the tumor tissues. Together, cabozantinib + Honokiol combination can significantly inhibit c-Met-induced and Nrf2-mediated anti-oxidant pathway in renal cancer cells to promote increased oxidative stress and tumor cell death.

Anticancer effects of ABTL0812, a clinical stage drug inducer of autophagy-mediated cancer cell death, in glioblastoma models.

Glioblastoma multiforme (GBM) is the most malignant adult brain tumor. Current standard of care treatments have very limited efficacy, being the patients´ overall survival 14 months and the 2-year survival rate less than 10%. Therefore, the treatment of GBM is an urgent unmet clinical need. The aim of this study was to investigate in vitro and in vivo the potential of ABTL0812, an oral anticancer compound currently in phase II clinical stage, as a novel therapy for GBM. We showed that ABTL0812 inhibits cell proliferation in a wide panel of GBM cell lines and patient-derived glioblastoma stem cells (GSCs) with half maximal inhibitory concentrations (IC50s) ranging from 15.2 µM to 46.9 µM. Additionally, ABTL0812 decreased GSCs neurosphere formation. GBM cells aggressiveness is associated with a trans-differentiation process towards a less differentiated phenotype known as proneural to mesenchymal transition (PMT). ABTL0812 was shown to revert PMT and induce cell differentiation to a less malignant phenotype in GBM cell lines and GSCs, and consequently reduced cell invasion. As previously shown in other cancer types, we demonstrated that the molecular mechanism of action of ABTL0812 in glioblastoma involves the inhibition of Akt/mTORC1 axis by overexpression of TRIB3, and the activation of endoplasmic reticulum (ER) stress/unfolded protein response (UPR). Both actions converge to induce autophagy-mediated cell death. ABTL0812 anticancer efficacy was studied in vivo using subcutaneous and orthotopic intra-brain xenograft tumor models. We demonstrated that ABTL0812 impairs tumor growth and increases disease-free survival and overall survival of mice. Furthermore, the histological analysis of tumors indicated that ABTL0812 decreases angiogenesis. Finally, we investigated the combination of ABTL0812 with the standard of care treatments for GBM radiotherapy and temozolomide in an orthotopic model, detecting that ABTL0812 potentiates the efficacy of both treatments and that the strongest effect is obtained with the triple combination of ABTL0812+radiotherapy+temozolomide. Overall, the present study demonstrated the anticancer efficacy of ABTL0812 as single agent and in combination with the GBM standard of care treatments in models of glioblastoma and supports the clinical investigation of ABTL0812 as a potential novel therapy for this aggressive brain tumor type.

ERK5 Inhibition Induces Autophagy-Mediated Cancer Cell Death by Activating ER Stress.

Autophagy is a highly conserved intracellular process that preserves cellular homeostasis by mediating the lysosomal degradation of virtually any component of the cytoplasm. Autophagy is a key instrument of cellular response to several stresses, including endoplasmic reticulum (ER) stress. Cancer cells have developed high dependency on autophagy to overcome the hostile tumor microenvironment. Thus, pharmacological activation or inhibition of autophagy is emerging as a novel antitumor strategy. ERK5 is a novel member of the MAP kinase family that is activated in response to growth factors and different forms of stress. Recent work has pointed ERK5 as a major player controlling cancer cell proliferation and survival. Therefore small-molecule inhibitors of ERK5 have shown promising therapeutic potential in different cancer models. Here, we report for the first time ERK5 as a negative regulator of autophagy. Thus, ERK5 inhibition or silencing induced autophagy in a panel of human cancer cell lines with different mutation patterns. As reported previously, ERK5 inhibitors (ERK5i) induced apoptotic cancer cell death. Importantly, we found that autophagy mediates the cytotoxic effect of ERK5i, since ATG5ˉ/ˉ autophagy-deficient cells viability was not affected by these compounds. Mechanistically, ERK5i stimulated autophagic flux independently of the canonical regulators AMPK or mTORC1. Moreover, ERK5 inhibition resulted in ER stress and activation of the Unfolded Protein Response (UPR) pathways. Specifically, ERK5i induced expression of the ER luminal chaperone BiP (a hallmark of ER stress), the UPR markers CHOP and ATF4, and the spliced form of XBP1. Pharmacological inhibition of UPR with chemical chaperone TUDC, or ATF4 silencing, resulted in impaired ERK5i-mediated UPR, autophagy and cytotoxicity. Overall, our results suggest that ERK5 inhibition induces autophagy-mediated cancer cell death by activating ER stress. Since ERK5 inhibition sensitizes cancer cells and tumors to chemotherapy, future work will determine the relevance of UPR and autophagy in the combined use of chemotherapy and ERK5i to tackle Cancer.

MA01.06 Phase 2 of Pro-Autophagic Drug ABTL0812 in Combination With First-Line Paclitaxel and Carboplatin in IIIb/IV Squamous NSCLC

ABTL0812 induces cytotoxic autophagy-mediated cancer cell death. It induces endoplasmic reticular (ER)-stress, and inhibits Akt/mTOR axis by upregulating TRIB3, an endogenous Akt inhibitor. Both actions, ER stress and blockade of Akt/mTOR, converge to induce a sustained and robust autophagy. Preclinical data in non-small cell lung carcinoma (NSCLC) indicated efficacy as a single agent and synergy with chemotherapy. A phase 1 of ABTL0812 in combination with paclitaxel and carboplatin (P/C) confirmed the safety of the triple combination

Challenges and Therapeutic Opportunities of Autophagy in Cancer Therapy.

Simple SummaryAutophagy is a physiological process characterized by the degradation of the cell components through lysosomes due to stimuli/stress. In this study, we review the challenges and therapeutic opportunities that autophagy presents in the treatment of cancer. We discussed the results of several studies that evaluated autophagy as a therapeutic strategy in cancer, both through the modulation of therapeutic resistance and the death of cancer cells. Moreover, we discussed the role of autophagy in the biology of cancer stem cells and the inhibition of this process as a strategy to overcome resistance and progression of cancer stem cells.Autophagy is a physiological cellular process that is crucial for development and can occurs in response to nutrient deprivation or metabolic disorders. Interestingly, autophagy plays a dual role in cancer cells—while in some situations, it has a cytoprotective effect that causes chemotherapy resistance, in others, it has a cytotoxic effect in which some compounds induce autophagy-mediated cell death. In this review, we summarize strategies aimed at autophagy for the treatment of cancer, including studies of drugs that can modulate autophagy-mediated resistance, and/or drugs that cause autophagy-mediated cancer cell death. In addition, the role of autophagy in the biology of cancer stem cells has also been discussed.

Abstract 1234: The anticancer drug ABTL0812 induces cancer cell death by impairing Akt/mTORC1 axis and inducing ER stress-mediated cytotoxic autophagy

Abstract ABTL0812 is a first-in-class small molecule with anticancer activity currently in Phase 2a clinical evaluation in patients with advanced endometrial and squamous NSCLC. We have previously described that ABTL0812 induces TRIB3 pseudokinase expression, resulting in inhibition of the Akt-mTORC1 axis and autophagy-mediated cancer cell death. However, classical PI3K/Akt/mTOR inhibitors do not induce an autophagy as strong as ABTL0812 does, therefore we aimed to further elucidate the molecular mechanism responsible for the cytotoxic autophagy which causes ABTL0812 anticancer activity. ABTL0812 induced UPR hallmarks ATF4, CHOP and TRIB3 in vitro in lung, endometrial and pancreatic cancer cell lines, as well as in non-tumor cells. Nevertheless, therapeutic concentrations of ABTL0812 did not induce cytotoxic autophagy in non-tumor cells. Furthermore, genetic or pharmacological inhibition of the UPR resulted in impaired ABTL0812 cytotoxicity in cancer cells. Expression of UPR markers (ATF4, CHOP and TRIB3) in response to ABTL0812 treatment were also validated in xenograft models. In order to uncover the precise molecular mechanism involved in ABTL0812-induced UPR and cytotoxic autophagy we undertook a comprehensive sphingolipidomic analysis, since changes in sphingolipids have been reported to contribute to activation of both UPR and autophagy. ABTL0812 treatment resulted in increased levels of long chain dihydroceramides in cultured cancer cells as well as in vivo. Mechanistically, ABTL0812 impaired desaturase-1 activity (Des-1), the enzyme that introduces a 4,5-trans-double bond in the sphingolipid backbone of dihydroceramides to generate ceramides. Accordingly, in vitro incubation of cancer cells with dihydroceramides resulted in activation of UPR, autophagy and cytotoxicity. Of interest, we observed that tumor cells showed higher Des-1 expression levels than non-tumor cells. Finally, we showed that Des-1 inhibition (GT11) and mTORC1 inhibition (everolimus) collaborate to promote autophagy and cancer cell death, simulating ABTL0812 activity. Furthermore, we have validated the increased expression of CHOP and TRIB3 mRNA levels in blood samples from ABTL0812 treated patients. These biomarkers are currently used as pharmacodynamic biomarkers in the ongoing phase 2 clinical trial in patients with squamous NSCLC and endometrial cancer. To our knowledge, this is the first time that UPR markers are reported to change in human blood in response to any drug treatment. In conclusion, we have shown that ABTL0812 triggers a sustained ER stress and UPR activation mediated by the impairment of Des-1 activity which collaborates with mTORC1 inhibition to induce cytotoxic autophagy in cancer cells, which offers improved anticancer activity over just inhibiting mTORC1. Citation Format: Pau Muñoz-Guardiola, Josefina Casas, Elisabet Megías-Roda, Hector Perez-Montoyo, Sonia Solé-Sánchez, Marc Yeste-Velasco, Tatiana Erazo, Nora Diéguez-Martínez, Sergio Espinosa-Gil, Guillermo Yoldi, Cristina Muñoz-Pinedo, Miguel F. Segura, Jose Alfon, Gemma Fabriàs, Guillermo Velasco, Carles Domenech, Jose M. Lizcano. The anticancer drug ABTL0812 induces cancer cell death by impairing Akt/mTORC1 axis and inducing ER stress-mediated cytotoxic autophagy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1234.

The anti-cancer drug ABTL0812 induces ER stress-mediated cytotoxic autophagy by increasing dihydroceramide levels in cancer cells

ABSTRACT ABTL0812 is a first-in-class small molecule with anti-cancer activity, which is currently in clinical evaluation in a phase 2 trial in patients with advanced endometrial and squamous non-small cell lung carcinoma (NCT03366480). Previously, we showed that ABTL0812 induces TRIB3 pseudokinase expression, resulting in the inhibition of the AKT-MTORC1 axis and macroautophagy/autophagy-mediated cancer cell death. However, the precise molecular determinants involved in the cytotoxic autophagy caused by ABTL0812 remained unclear. Using a wide range of biochemical and lipidomic analyses, we demonstrated that ABTL0812 increases cellular long-chain dihydroceramides by impairing DEGS1 (delta 4-desaturase, sphingolipid 1) activity, which resulted in sustained ER stress and activated unfolded protein response (UPR) via ATF4-DDIT3-TRIB3 that ultimately promotes cytotoxic autophagy in cancer cells. Accordingly, pharmacological manipulation to increase cellular dihydroceramides or incubation with exogenous dihydroceramides resulted in ER stress, UPR and autophagy-mediated cancer cell death. Importantly, we have optimized a method to quantify mRNAs in blood samples from patients enrolled in the ongoing clinical trial, who showed significant increased DDIT3 and TRIB3 mRNAs. This is the first time that UPR markers are reported to change in human blood in response to any drug treatment, supporting their use as pharmacodynamic biomarkers for compounds that activate ER stress in humans. Finally, we found that MTORC1 inhibition and dihydroceramide accumulation synergized to induce autophagy and cytotoxicity, phenocopying the effect of ABTL0812. Given the fact that ABTL0812 is under clinical development, our findings support the hypothesis that manipulation of dihydroceramide levels might represents a new therapeutic strategy to target cancer. Abbreviations: 4-PBA: 4-phenylbutyrate; AKT: AKT serine/threonine kinase; ATG: autophagy related; ATF4: activating transcription factor 4; Cer: ceramide; DDIT3: DNA damage inducible transcript 3; DEGS1: delta 4-desaturase, sphingolipid 1; dhCer: dihydroceramide; EIF2A: eukaryotic translation initiation factor 2 alpha; EIF2AK3: eukaryotic translation initiation factor 2 alpha kinase 3; ER: endoplasmic reticulum; HSPA5: heat shock protein family A (Hsp70) member 5; MAP1LC3B: microtubule associated protein 1 light chain 3 beta; MEF: mouse embryonic fibroblast; MTORC1: mechanistic target of rapamycin kinase complex 1; NSCLC: non-small cell lung cancer; THC: Δ9-tetrahydrocannabinol; TRIB3: tribbles pseudokinase 3; XBP1: X-box binding protein 1; UPR: unfolded protein response.

Inhibiting SUMO1-mediated SUMOylation induces autophagy-mediated cancer cell death and reduces tumour cell invasion via RAC1.

ABSTRACTPost-translational modifications directly control protein activity and, thus, they represent an important means to regulate the responses of cells to different stimuli. Protein SUMOylation has recently been recognised as one such modification, and it has been associated with various diseases, including different types of cancer. However, the precise way that changes in SUMOylation influence the tumorigenic properties of cells remains to be fully clarified. Here, we show that blocking the SUMO pathway by depleting SUMO1 and UBC9, or by exposure to ginkgolic acid C15:1 or 2-D08 (two different SUMOylation inhibitors), induces cell death, also inhibiting the invasiveness of tumour cells. Indeed, diminishing the formation of SUMO1 complexes induces autophagy-mediated cancer cell death through increasing the expression of Tribbles pseudokinase 3 (TRIB3). Moreover, we found that blocking the SUMO pathway inhibits tumour cell invasion by decreasing RAC1 SUMOylation. These findings shed new light on the mechanisms by which SUMO1 modifications regulate the survival, and the migratory and invasive capacity of tumour cells, potentially establishing the bases to develop novel anti-cancer treatments based on the inhibition of SUMOylation.

Abstract 359: Dual BET/kinase inhibitors as a novel strategy for the treatment of multiple myeloma

Abstract The bromodomain and extra-terminal (BET) family of proteins are important epigenetic regulators of critical oncogenes, including c-Myc. JQ1 is the most potent BRD4 inhibitor, currently used in several clinical trials and effective in preventing multiple myeloma progression in vivo. Currently, clinical data is showing that patients often develop acquired resistance, indicating that single agent therapies targeting BRD4 such as JQ1 may not provide durable therapeutic responses. To overcome this limitation, we have developed a new class of dual inhibitors, capable to simultaneously target BRD4 and a panel of tyrosine kinases highly expressed in cancer including JAK2, FLT3, RET, FGFR1, ULK1 and ULK3. A preliminary in vitro screening identified SG3-014 as the lead compound, showing BRD4 and c-Myc inhibition activity similar to JQ1, but additionally, it is capable of inhibiting kinases that are over-activated in cancer and responsible for cytokine-mediated drug resistance pathways. In vitro assays on MM cell lines reveals higher SG3-014 sensitivity (5TGM1, 0.85μM; U266, 0.99μM) compared to JQ1 (5TGM1, 4.7μM; U266, 16μM). In vivo studies using 1x106 5TGM1-Luc cells demonstrate that SG3-014 treatment (25mg/Kg) significantly contributes to overall survival compared to JQ1 and the vehicle control cohort (n=10/group) (median survival CTRL=40.5; SG3-014=50.5; JQ1=46 days). Post-study analyses demonstrated a significant reduction in myeloma induced bone disease in the SG3-014 treated mice as measured by X-ray/ μCT/ histomorphometry. Clinical datasets identified ULK3 as the candidate kinase of SG3-014 targeting activity and correlated its expression with MM disease stages. Since ULK3 is involved in cellular senescence and autophagy regulation, a key mechanism by which several cancer cell lines protect themselves against apoptosis and become resistant to standard treatments, we explored the role of SG3-014 inhibition in the autophagy pathway. Preliminary data confirm that SG3-014 is an autophagy inhibitor and, as opposed to JQ1, downregulates ATG proteins level, leading to dysfunctional autophagosome formation. We noted that within 6 h, SG3-014 completely shut down autophagy with decrease levels of ULK1, beclin-1, ATG16L-1, ATG12, ATG3, p62 and increased LC3I/II ratios (additionally confirmed by microscopy and flow cytometry). These effects were not noted with JQ1. Further investigation is clarifying the mechanism by which SG3-014 inhibitory activity on ULK3 is responsible for autophagy mediated cancer cell death. In order to validate this target in the progression of the human disease, our future directions will analyze ex vivo multiple myeloma patient samples and examine the efficacy of the inhibitor in CD138 isolated multiple myeloma cells derived from patient biopsies. We propose dual BRD4/autophagy inhibition as a resilient strategy to prevent multiple myeloma by-pass of single targeted therapies. Citation Format: Marilena Tauro, Muhammad Ayaz, Harshani R. Lawrence, Nicholas J. Lawrence, Kenneth H. Shain, Ernst Schonbrunn, Conor C. Lynch. Dual BET/kinase inhibitors as a novel strategy for the treatment of multiple myeloma [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 359.

The role of autophagy and cancer

Autophagy is the main cellular degradation pathway for the clearance of damaged or superfluous proteins and organelles and represents the principle catabolic process regulating cellular homeostasis and organelle and protein turnover. Besides its role in cellular homeostasis, autophagy can be a form of programmed cell death or play a cytoprotective role, for example in situations of nutrient starvation. Accordingly, autophagy plays a dual role in cancer as this cellular process may help to overcome the stress evoked at the initial steps of tumorigenesis or work as a tumor suppressor mechanism. Moreover, different anticancer treatments activate autophagy in tumor cells, which either enhance cancer cell death or act as a mechanism of resistance to chemotherapy. Prior work by our group showed that Δ9-tetrahydrocannabinol (THC, the main active component of marijuana) triggers autophagy-mediated cancer cell death. In this presentation I will summarize several recent findings supporting that the modification of the sphingolipid metabolism of cancer cells by cannabinoids plays a pivotal role in the stimulation of autophagy-mediated cancer cell death.

Dihydroceramide accumulation mediates cytotoxic autophagy of cancer cells via autolysosome destabilization

ABSTRACTAutophagy is considered primarily a cell survival process, although it can also lead to cell death. However, the factors that dictate the shift between these 2 opposite outcomes remain largely unknown. In this work, we used Δ9-tetrahydrocannabinol (THC, the main active component of marijuana, a compound that triggers autophagy-mediated cancer cell death) and nutrient deprivation (an autophagic stimulus that triggers cytoprotective autophagy) to investigate the precise molecular mechanisms responsible for the activation of cytotoxic autophagy in cancer cells. By using a wide array of experimental approaches we show that THC (but not nutrient deprivation) increases the dihydroceramide:ceramide ratio in the endoplasmic reticulum of glioma cells, and this alteration is directed to autophagosomes and autolysosomes to promote lysosomal membrane permeabilization, cathepsin release and the subsequent activation of apoptotic cell death. These findings pave the way to clarify the regulatory mechanisms that determine the selective activation of autophagy-mediated cancer cell death.

The use of cannabinoids as anticancer agents

It is well-established that cannabinoids exert palliative effects on some cancer-associated symptoms. In addition evidences obtained during the last fifteen years support that these compounds can reduce tumor growth in animal models of cancer. Cannabinoids have been shown to activate an ER-stress related pathway that leads to the stimulation of autophagy-mediated cancer cell death. In addition, cannabinoids inhibit tumor angiogenesis and decrease cancer cell migration. The mechanisms of resistance to cannabinoid anticancer action as well as the possible strategies to develop cannabinoid-based combinational therapies to fight cancer have also started to be explored. In this review we will summarize these observations (that have already helped to set the bases for the development of the first clinical studies to investigate the potential clinical benefit of using cannabinoids in anticancer therapies) and will discuss the possible future avenues of research in this area.