Autophagic Cancer Cell Research Articles

β-Carboline-based light and pH dual stimuli-responsive ion transporters induce cancer cell death.

Light and pH dual-responsive ion transporters offer better applicability for cancer due to higher tunability and low cytotoxicity. Herein, we demonstrate the development of pH-responsive β-carboline-based ionophores and photocleavable-linker appended β-carboline-based proionophores to facilitate the controlled transport of Cl- across membranes, leading to apoptotic and autophagic cancer cell death.

DIRAS3: An Imprinted Tumor Suppressor Gene that Regulates RAS and PI3K-driven Cancer Growth, Motility, Autophagy, and Tumor Dormancy.

DIRAS3 is an imprinted tumor suppressor gene that encodes a 26 kDa GTPase with 60% amino acid homology to RAS, but with a distinctive 34 amino acid N-terminal extension required to block RAS function. DIRAS3 is maternally imprinted and expressed only from the paternal allele in normal cells. Loss of expression can occur in a single "hit" through multiple mechanisms. Downregulation of DIRAS3 occurs in cancers of the ovary, breast, lung, prostate, colon, brain, and thyroid. Reexpression of DIRAS3 inhibits signaling through PI3 kinase/AKT, JAK/STAT, and RAS/MAPK, blocking malignant transformation, inhibiting cancer cell growth and motility, and preventing angiogenesis. DIRAS3 is a unique endogenous RAS inhibitor that binds directly to RAS, disrupting RAS dimers and clusters, and preventing RAS-induced transformation. DIRAS3 is essential for autophagy and triggers this process through multiple mechanisms. Reexpression of DIRAS3 induces dormancy in a nu/nu mouse xenograft model of ovarian cancer, inhibiting cancer cell growth and angiogenesis. DIRAS3-mediated induction of autophagy facilitates the survival of dormant cancer cells in a nutrient-poor environment. DIRAS3 expression in dormant, drug-resistant autophagic cancer cells can serve as a biomarker and as a target for novel therapy to eliminate the residual disease that remains after conventional therapy.

Suppression of DYRK1A/B Drives Endoplasmic Reticulum Stress-mediated Autophagic Cell Death Through Metabolic Reprogramming in Colorectal Cancer Cells.

We previously identified KS40008 (4-(3-(4-hydroxyphenyl)-1H-pyrazolo[3,4-b]pyridin-5-yl)benzene-1,2-diol), a novel inhibitor of dual-specificity tyrosine phosphorylation-regulated kinase family (DYRK) 1A/B, which exhibited high enzymatic activity and cell proliferation-inhibitory effects in colorectal cancer (CRC) cell lines. In the present study, we aimed to elucidate the antitumor mechanisms of KS40008. To assess the cytotoxicity of KS40008, we utilized a human cell line and organoid model and performed a CCK-8 assay and real-time cell analysis. Mitochondrial function was determined through mitochondrial staining, mito-stress test, and glycolysis test. In addition, we investigated the mechanisms of cancer cell death induced by KS40008 through immunoblotting, real-time quantitative polymerase chain reaction, reactive oxygen species staining, and immunofluorescence staining. KS40008 exhibited significant cytotoxicity in CRC and non-CRC cell lines, and organoid models compared to 5-fluorouracil, a conventional chemotherapeutic drug. Moreover, KS40008-induced inhibition of DYRK1A/B led to mitochondrial dysfunction and endoplasmic reticulum stress, promoting autophagic cancer cell death. KS40008 exerts antitumor activity through the inhibition of DYRK1A/B. Here, we demonstrated a mechanism by which KS40008 affects endoplasmic reticulum stress-mediated autophagy through the induction of mitochondrial stress, leading to cytotoxicity in CRC.

Biochemical studies of the effect of arsenic trioxide on Ehrlich ascites tumor: Thesis Abstract

Thesis Abstract Hexokinase 2 enzyme catalyzes the rate-limiting step in glycolysis. It is overexpressed in several carcinomas including breast cancer to sustain energy for rapidly dividing cells and associates with chemoresistance. However, impact of chemo drugs alone or in combination on hexokinase activity and autophagic cell death is unclear. Arsenic trioxide (As2O3) (ATO) is a metalloid with potent antineoplastic effects in several types of cancer especially acute promyelocytic leukemia and breast cancer. Due to the cytotoxicity and low efficacy of ATO when it is used alone, a combination of ATO with other chemotherapeutic drugs may provide a rational basis for novel therapeutic combinations. Despite these efforts, the specific cellular events that account for differential effects of ATO on cancer cells’viability and the possible therapeutic combinations remain to be well‐defined. In this report, we used an in vivo murine adenocarcinoma model to validate the effects of As2O3 and cisplatin on hexokinase activity and autophagic cancer cell death. We found that the two drugs inhibit hexokinase activity and induceautophagic marker, beclin 1 expression. Interestingly, combining As2O3 with cisplatin synergistically enhanced these effects and alleviated oxidative stress often encountered in As2O3 treatment. Altogether, our data provide direct evidence that inhibition of hexokinase activity and induction of autophagic cell death are mediating the antineoplastic effects of As2O3 and cisplatin. Our findings raise the potential of combiningAs2O3 with cisplatin as an approach to augment cisplatin-induced cell death and combat cisplatin chemo-resistance in cancer.

Autophagy promotes oncolysis of an adenovirus expressing apoptin in human bladder cancer models.

As a potential cancer therapy, we developed a recombinant adenovirus named Ad-VT, which was designed to express the apoptosis-inducing gene (apoptin) and selectively replicate in cancer cells via E1a manipulation. However, how it performs in bladder cancer remains unclear. We examined the antitumor efficacy of Ad-VT in bladder cancers using CCK-8 assays and xenograft models. Autophagy levels were evaluated by western blotting, MDC staining, and RFP-GFP-LC3 aggregates' analyses. Here, we report the selective replication and antitumor efficacy (viability inhibition and apoptosis induction) of Ad-VT in bladder cancer cells. Using xenograft tumor models, we demonstrate that its effects are tumor specific resulting in the inhibition of tumor growth and improvement of the survival of mice models. Most Importantly, Ad-VT induced a complete autophagy flux leading to autophagic cancer cell death through a signaling pathway involving AMPK, raptor and mTOR. Finally, we suggest that treatment combination of Ad-VT and rapamycin results in a synergistic improvement of tumor control and survival compared to monotherapy. This study suggests that Ad-VT can induce selective autophagic antitumor activities in bladder cancer through the AMPK-Raptor-mTOR pathway, which can be further improved by rapamycin.

Abstract 4083: Crizotinib improves the therapeutic efficacy of olaparib in ovarian cancer

Abstract Despite improvements in treatment, ovarian cancer still proves lethal in >60% of cases. One of the most important factors contributing to poor patient outcomes is the ability of dormant ovarian cancer cells to survive following primary surgery and chemotherapy. Poly (ADP) ribose polymerase inhibitors (PARPi) are promising agents for treating both primary and recurrent ovarian cancers. However, acquired drug resistance to PARPi decreases the clinical efficacy of these agents. We have previously found that olaparib induces autophagy in ovarian cancer and the addition of drugs that inhibit autophagy, such as chloroquine, can overcome drug resistance and enhance the anti-cancer activity of PARP inhibitors in ovarian cancer cells, cell line-derived xenograft and patient-derived xenograft models. Building upon this work, our group has sought drugs that are selectively toxic for autophagic ovarian cancer cells and that might enhance sensitivity to PARP inhibitors. In a previous study, our laboratory had found that knockdown of the anaplastic lymphoma kinase (ALK) significantly reduced survival of ovarian cancer cells that were undergoing autophagy. The FDA-approved ALK inhibitor, crizotinib, exhibited significantly greater toxicity for autophagic ovarian cancer cells in culture and cured a fraction of mice with dormant, autophagic ovarian cancer xenografts. As olaparib induces autophagy in ovarian cancer cells and crizotinib selectively targets autophagic cancer cells, we 1) explored the vulnerability of autophagic cells to the combination treatment of olaparib and crizotinib both in vitro and in vivo; and 2) the mechanism by which the combination of olaparib and crizotinib regulates cell proliferation and programmed cell death. Enhancement of olaparib activity was observed in 4 ovarian cancer cell lines (OVCAR8, OVCAR3, HEY and SKOV3). Moreover, a combination of olaparib and crizotinib proved more effective than either single agent in a xenograft model of the OVCAR8 ovarian cancer cell line. Mechanistically, a combination of olaparib and crizotinib induces DNA damage and accumulation of γ-H2AX. A combination of olaparib and crizotinib increased reactive oxygen species, while decreasing the phosphorylation of AKT. This decrease in p-AKT was associated with a decrease in p-mTOR, all of which contributed to an increase in autophagy, documented by western blot analysis, LC3 puncta and electron microscopy. Decreased p-AKT also correlated with a decrease in its downstream effector, survivin, leading to apoptosis. Together, our data suggest that crizotinib can enhance the therapeutic efficacy of olaparib in ovarian cancer by modulating autophagy and apoptosis. Moreover, we have identified a unique combination of therapy with olaparib and crizotinib that could improve the therapeutic efficacy of olaparib and outcomes for ovarian cancer patients. Citation Format: Janice M. Santiago-O'Farrill, Alicia M. Blessing, Sarah E. Becker, Zhen Lu, Robert C. Bast. Crizotinib improves the therapeutic efficacy of olaparib in ovarian cancer [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 4083.

Elimination of dormant, autophagic ovarian cancer cells and xenografts through enhanced sensitivity to anaplastic lymphoma kinase inhibition.

Poor outcomes for patients with ovarian cancer relate to dormant, drug-resistant cancer cells that survive after primary surgery and chemotherapy. Ovarian cancer (OvCa) cells persist in poorly vascularized scars on the peritoneal surface and depend on autophagy to survive nutrient deprivation. The authors have sought drugs that target autophagic cancer cells selectively to eliminate residual disease. By using unbiased small-interfering RNA (siRNA) screens, the authors observed that knockdown of anaplastic lymphoma kinase (ALK) reduced the survival of autophagic OvCa cells. Small-molecule ALK inhibitors were evaluated for their selective toxicity against autophagic OvCa cell lines and xenografts. Autophagy was induced by reexpression of GTP-binding protein Di-Ras3 (DIRAS3) or serum starvation and was evaluated with Western blot analysis, fluorescence imaging, and transmission electron microscopy. Signaling pathways required for crizotinib-induced apoptosis of autophagic cells were explored with flow cytometric analysis, Western blot analysis, short-hairpin RNA knockdown of autophagic proteins, and small-molecule inhibitors of STAT3 and BCL-2. Induction of autophagy by reexpression of DIRAS3 or serum starvation in multiple OvCa cell lines significantly reduced the 50% inhibitory concentration of crizotinib and other ALK inhibitors. In 2 human OvCa xenograft models, the DIRAS3-expressing tumors treated with crizotinib had significantly decreased tumor burden and long-term survival in 67% to 79% of mice. Crizotinib treatment of autophagic cancer cells further enhanced autophagy and induced autophagy-mediated apoptosis by decreasing phosphorylated STAT3 and BCL-2 signaling. Crizotinib may eliminate dormant, autophagic, drug-resistant OvCa cells that remain after conventional cytoreductive surgery and combination chemotherapy. A clinical trial of ALK inhibitors as maintenance therapy after second-look operations should be seriously considered.

Light-triggered selective ROS-dependent autophagy by bioactive nanoliposomes for efficient cancer theranostics.

Light-responsive nanoliposomes are being reported to induce cancer cell death through heat and reactive oxygen species (ROS). Nanoliposomes (CIR NLPs) encapsulating a near-infrared (NIR) light-sensitive dye, IR780, and a bioactive chlorophyll-rich fraction of Anthocephalus cadamba (CfAc) were synthesized and characterized. These CIR NLPs, when activated by NIR light, displayed localized synergistic cancer cell death under in vitro and in vivo conditions. We demonstrated a NIR light-mediated release of CfAc in cancer cells. The bioactive CfAc was selective in causing ROS generation (leading to autophagic cell death) in cancer cells, while normal healthy cells were unaffected. An increase in the intracellular ROS leading to enhanced lipidation of microtubule-associated protein light chain 3 (LC3-II) was observed only in cancer cells, while normal cells showed no increase in either ROS or LC3-II. In vivo analysis of CIR NLPs in an orthotopic mouse model showed better anti-tumorigenic potential through a combined effect (i.e. via heat and CfAc). We reported for the first time induction of selective and localized, bioactive phyto fraction-mediated autophagic cancer cell death through an NIR light trigger. The synergistic activation of ROS-mediated autophagy by light-triggered nanoliposomes can be a useful strategy for enhancing the anticancer potential of combinational therapies.

Abstract 4263: Autophagic ovarian cancer cells exhibit substantially enhanced sensitivity to ALK inhibition

Abstract One of the major factors contributing to poor outcomes for patients with ovarian cancer is the persistence of dormant, drug resistant cancer cells after primary surgery and chemotherapy. The persistent cancer found in positive second look operations is poorly vascularized and autophagy is widespread in more than 80% of cases. Consequently, drugs that regulate survival in autophagic cancer cells may be much more active when administered as maintenance therapy than when used to treat gross recurrent disease. Using unbiased siRNA screens, we have identified target genes that regulate the survival of ovarian cancer cells that are undergoing autophagy-induced by the re-expression of DIRAS3 (ARHI) or by serum starvation. Knockdown of the anaplastic lymphoma kinase (ALK) significantly reduced survival of ovarian cancer cells that were undergoing autophagy. Importantly, FDA-approved ALK inhibitors, including crizotinib, exhibited significantly greater toxicity after induction of autophagy by upregulation of DIRAS3 or serum starvation in 5 ovarian cancer cell lines. Induction of autophagy by upregulation of DIRAS3 or serum starvation in ovarian cancer cells reduced the IC50 of crizotinib and other ALK inhibitors ranging from 1.5 to 20-fold (all p-values <0.05). Crizotinib treatment in autophagic cancer cells enhanced autophagy and apoptosis via a decrease in p-STAT3 and BCL-2 signaling. Selective targeting of p-STAT3 or BCL-2 also resulted in significantly greater toxicity after induction of autophagy by upregulation of DIRAS3 or serum starvation in multiple ovarian cancer cells. Inducible upregulation of DIRAS3 by doxycycline in the first 6 weeks prompted autophagy and dormancy in two separate xenograft models (SKOV3 and OVCAR8). Crizotinib treatment of dormant autophagic SKOV3 xenografts prolonged median survival by 9.7 weeks (p<0.0032) and cured a subset of mice. Thirteen of fifteen mice (87%) bearing SKOV3 xenografts and nine of eleven mice (82%) bearing OVCAR8 xenografts, were tumor free at 200 days compared to the other three control arms (overall survival, p<0.0001 and p<0.05, respectively). Our studies suggest that ALK inhibitors might provide an effective agent to eliminate autophagic, dormant, drug resistant ovarian cancer cells that remain after conventional cytoreductive surgery and combination chemotherapy. Citation Format: Alicia M. Blessing, Weiqun Mao, Lan Pang, Philip Rask, Zhen Lu, Robert C. Bast. Autophagic ovarian cancer cells exhibit substantially enhanced sensitivity to ALK inhibition [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 4263.

Combination of arsenic trioxide and cisplatin synergistically inhibits both hexokinase activity and viability of Ehrlich ascites carcinoma cells.

Hexokinase-2 is overexpressed in several carcinomas including breast cancer to sustain energy for rapidly dividing cells and associates with chemoresistance. However, the impact of chemo drugs (alone or in combination) on hexokinase activity and autophagic cell death is unclear. In this report, we used an in vivo murine adenocarcinoma model to validate the effects of As2 O3 and cisplatin on hexokinase activity and autophagic cancer cell death. We found that the two drugs inhibit hexokinase activity and induce autophagic marker, beclin 1 expression. Interestingly, combining As2 O3 with cisplatin synergistically enhanced these effects and alleviated oxidative stress often encountered in As2 O3 treatment. Altogether, our data provide direct evidence that inhibition of hexokinase activity and induction of autophagic cell death are mediating the antineoplastic effects of As2 O3 and cisplatin. Our findings raise the potential of combining As2 O3 with cisplatin as an approach to augment cisplatin-induced cell death and combat cisplatin chemoresistance in cancer.

High-selective HDAC6 inhibitor promotes HDAC6 degradation following autophagy modulation and enhanced antitumor immunity in glioblastoma

Glioblastoma is the most fatal type of primary brain cancer, and current treatments for glioblastoma are insufficient. HDAC6 is overexpressed in glioblastoma, and siRNA-mediated knockdown of HDAC6 inhibits glioma cell proliferation. Herein, we report a high-selective HDAC6 inhibitor, J22352, which has PROTAC (proteolysis-targeting chimeras)-like property resulted in both p62 accumulation and proteasomal degradation, leading to proteolysis of aberrantly overexpressed HDAC6 in glioblastoma. The consequences of decreased HDAC6 expression in response to J22352 decreased cell migration, increased autophagic cancer cell death and significant tumor growth inhibition. Notably, J22352 reduced the immunosuppressive activity of PD-L1, leading to the restoration of host anti-tumor activity. These results demonstrate that J22352 promotes HDAC6 degradation and induces anticancer effects by inhibiting autophagy and eliciting the antitumor immune response in glioblastoma. Therefore, this highly selective HDAC6 inhibitor can be considered a potential therapeutic for the treatment of glioblastoma and other cancers.

A research update on the anticancer effects of bufalin and its derivatives.

Bufalin (BF) is a cardiotonic steroid that has recently been found to have substantial anticancer activity; however, more efforts should be directed toward clarifying the detailed molecular mechanisms underlying this activity. BF could exert its anticancer effect by inducing apoptosis in various human cancer cells and thus triggering autophagic cancer cell death. The anti-inflammatory activities of BF are potentially important for its anticancer functions. Notably, some promising synthetic BF derivatives, including poly (ethylene glycol)-based polymeric prodrug of BF and BF211, have shown potent anticancer activity. Additionally, clinical trials regarding the use of BF-related agents in patients have supported the positive effect of BF as an anticancer treatment. Currently, large-scale randomized, double-blind, placebo or positive drug parallel controlled studies are required to confirm the anticancer potential of BF in various cancer types in the clinical setting. The present review will evaluate the potential mechanisms mediated by BF in intracellular signaling events in cancer cells and various promising BF derivatives that may have greater anticancer activity, thereby clarifying BF-mediated anticancer effects. The experimental and clinical results reviewed strongly emphasize the importance of this topic in future investigations.

The role of vascular endothelial growth factor, interleukin 8, and insulinlike growth factor in sustaining autophagic DIRAS3-induced dormant ovarian cancer xenografts.

Re-expression of the imprinted tumor suppressor gene DIRAS family GTPase 3 (DIRAS3) (aplysia ras homology member I [ARHI]) induces autophagy and tumor dormancy in ovarian cancer xenografts, but drives autophagic cancer cell death in cell culture. The current study explored the tumor and host factors required to prevent autophagic cancer cell death in xenografts and the use of antibodies against those factors or their receptors to eliminate dormant autophagic ovarian cancer cells. Survival factors (insulinlike growth factor 1 [IGF-1], vascular endothelial growth factor [VEGF], and interleukin 8 [IL-8]) were detected with growth factor arrays and measured using enzyme-linked immunoadsorbent assay analysis. Phosphorylation of protein kinase B (AKT), phosphorylation of extracellular signal-regulated kinase (ERK), nuclear localization of translocation factor EB (TFEB) or forkhead box O3a (FOXo3a), and expression of microtubule-associated proteins 1A/1B light chain 3B (MAPLC3B; LC3B) were examined using Western blot analysis. The effect of treatment with antibodies against survival factors or their receptors was studied using DIRAS3-induced dormant xenograft models. Ovarian cancer cells grown subcutaneously in nude mice exhibited higher levels of phosphorylated ERK/AKT activity and lower levels of nuclear TFEB/FOXo3a, MAPLC3B, and autophagy compared with cells grown in culture. Induction of autophagy and dormancy with DIRAS3 was associated with decreased ERK/AKT signaling. The addition of VEGF, IGF-1, and IL-8 weakened the inhibitory effect of DIRAS3 on ERK/AKT activity and reduced DIRAS3-mediated TFEB or FOXo3a nuclear localization and MAPLC3B expression in ovarian cancer cells. Treatment with antibodies against VEGF, IL-8, and IGF receptor inhibited the growth of dormant xenografts, thereby prolonging survival from 99 to >220 days (P<.05) and curing a percentage of mice. Treatment with a combination of anti-VEGF, anti-IL-8, and anti-IGF receptor antibodies prevented the outgrowth of dormant cells and prolonged survival in a preclinical model.

RAS-related GTPases DIRAS1 and DIRAS2 induce autophagic cancer cell death and are required for autophagy in murine ovarian cancer cells

ABSTRACTAmong the 3 GTPases in the DIRAS family, DIRAS3/ARHI is the best characterized. DIRAS3 is an imprinted tumor suppressor gene that encodes a 26-kDa GTPase that shares 60% homology to RAS and RAP. DIRAS3 is downregulated in many tumor types, including ovarian cancer, where re-expression inhibits cancer cell growth, reduces motility, promotes tumor dormancy and induces macroautophagy/autophagy. Previously, we demonstrated that DIRAS3 is required for autophagy in human cells. Diras3 has been lost from the mouse genome during evolutionary re-arrangement, but murine cells can still undergo autophagy. We have tested whether DIRAS1 and DIRAS2, which are homologs found in both human and murine cells, could serve as surrogates to DIRAS3 in the murine genome affecting autophagy and cancer cell growth. Similar to DIRAS3, these 2 GTPases share 40–50% homology to RAS and RAP, but differ from DIRAS3 primarily in the lengths of their N-terminal extensions. We found that DIRAS1 and DIRAS2 are downregulated in ovarian cancer and are associated with decreased disease-free and overall survival. Re-expression of these genes suppressed growth of human and murine ovarian cancer cells by inducing autophagy-mediated cell death. Mechanistically, DIRAS1 and DIRAS2 induce and regulate autophagy by inhibition of the AKT1-MTOR and RAS-MAPK signaling pathways and modulating nuclear localization of the autophagy-related transcription factors FOXO3/FOXO3A and TFEB. Taken together, these data suggest that DIRAS1 and DIRAS2 likely serve as surrogates in the murine genome for DIRAS3, and may function as a backup system to fine-tune autophagy in humans.

Synergistic promoting effects of pentoxifylline and simvastatin on the apoptosis of triple-negative MDA-MB-231 breast cancer cells.

Pentoxifylline (PTX), a xanthine family molecule and simvastatin (SIM), an anti-hypercholesterolemic agent, have recently been considered as sensitizers to chemotherapy and radiotherapy. The present in vitro study evaluated their antitumor synergistic effects on MDA‑MB‑231 breast cancer cells characterized by the triple‑negative phenotype(TNP). The anti-proliferative effects of these two agents were evaluated by MTT and clonogenic assays. Cell cycle progression was examined using propidium iodide staining. Apoptosis was investigated by AnnexinV labeling, and by examining caspase3 activity and DNA fragmentation. Autophagic vesicles and reactive oxygen species (ROS) levels were monitored by flow cytometry. Western blot analysis was performed to evaluate molecular targets. Our results revealed that when used alone, PTX and SIM exerted antitumor effects. Nevertheless, used in combination, the inhibition of cell proliferation was synergistically superior (80% vs 42%) than that observed following treatment with each agent alone after 48h. PTX alone (0.5mM) induced both apoptosis (25%) and autophagy(25%); however, when used in combination with SIM (0.5µM), the balance between these processes was disrupted and the cells underwent apoptosis (>65%) as opposed to autophagy (<13%). This imbalance was associated with an increase in ERK1/2 and AKT activation, but not with an increase in mTOR phosphorylation, and with the suppression of the NF-κB pathway. In addition, in the cells treated with both agents, almost 78% of the cells were arrested at the G0/G1 phase and lost their colony-forming ability (38±5%) compared to the cells treated with PTX alone (115±5%). On the whole, these results suggest that the induction of autophagy may be a protective mechanism preventing MDA‑MB‑231 cancer cell death. The combined use of PTX and SIM may drive dormant autophagic cancer cells to undergo apoptosis and thus this may be a novel treatment strategy for breast cancer characterized by the TNP.

Abstract 3313: Autophagic ovarian cancer cells exhibit substantially enhanced sensitivity to Crizotinib

Abstract One of the major factors contributing to poor outcomes for patients with ovarian cancer is the persistence of dormant, drug resistant cancer cells after primary surgery and chemotherapy. Recurrent, progressively growing ovarian cancer metastases are generally well vascularized and may only have a small fraction of cancer cells undergoing autophagy. By contrast, the persistent, drug resistant, dormant cancer cells that remain on the peritoneal cavity after conventional treatment tend to be poorly vascularized. In this nutrient-poor, avascular microenvironment autophagy is widespread and can be found in more than 80% of cases. Drugs that regulate survival in autophagic cancer cells may be much more active when administered as maintenance therapy than when used to treat gross primary or recurrent disease. Using unbiased siRNA screens, we have identified target genes that regulate the survival of ovarian cancer cells that are undergoing autophagy that has been induced by the re-expression of DIRAS3 or by amino acid starvation. Knockdown of the anaplastic lymphoma kinase (ALK) significantly reduced survival of ovarian cancer cells that were undergoing autophagy. Importantly, the FDA-approved ALK inhibitor, Crizotinib, exhibited significantly greater toxicity after induction of autophagy by upregulation of DIRAS3 (ARHI) in OVCAR8, CAOV3, and SKOv3 ovarian cancer cells. Induction of autophagy by upregulation of DIRAS3 in SKOv3 ovarian cancer cells reduced the IC50 of Crizotinib from 7.44µM ± 0.04708 to 2.129µM ± 0.06152 (P&amp;lt;0.05). Conversely, siRNA knockdown of DIRAS3 in CAOV3 cells with endogenously high DIRAS3 expression and baseline autophagy increased the IC50 of Crizotinib from 204 nM ± 0.01265 to 267 nM ± 0.01715 (P&amp;lt;0.05). Our studies in cell culture suggest that Crizotinib might provide an effective agent to eliminate autophagic, dormant drug resistant ovarian cancer cells that remain after conventional cytoreductive surgery and combination chemotherapy. Subsequent studies will evaluate the effect of Crizotinib in xenograft models of DIRAS3-induced ovarian cancer dormancy. Citation Format: Alicia M. Blessing, Yan Wang, Weiqun Mao, Lan Pang, Zhen Lu, Robert C. Bast. Autophagic ovarian cancer cells exhibit substantially enhanced sensitivity to Crizotinib [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3313. doi:10.1158/1538-7445.AM2017-3313

W09, a novel autophagy enhancer, induces autophagy-dependent cell apoptosis via activation of the EGFR-mediated RAS-RAF1-MAP2K-MAPK1/3 pathway

ABSTRACTThe EGFR (epidermal growth factor receptor) signaling pathway is frequently deregulated in many malignancies. Therefore, targeting the EGFR pathway is regarded as a promising strategy for anticancer drug discovery. Herein, we identified a 2-amino-nicotinonitrile compound (w09) as a novel autophagy enhancer, which potently induced macroautophagy/autophagy and consequent apoptosis in gastric cancer cells. Mechanistic studies revealed that EGFR-mediated activation of the RAS-RAF1-MAP2K-MAPK1/3 signaling pathway played a critical role in w09-induced autophagy and apoptosis of gastric cancer cells. Inhibition of the MAPK1/3 pathway with U0126 or blockade of autophagy by specific chemical inhibitors markedly attenuated the effect of w09-mediated growth inhibition and caspase-dependent apoptosis. Furthermore, these conclusions were supported by knockdown of ATG5 or knockout of ATG5 and/or ATG7. Notably, w09 increased the expression of SQSTM1 by transcription, and knockout of SQSTM1 or deleting the LC3-interaction region domain of SQSTM1, significantly inhibited w09-induced PARP1 cleavage, suggesting the central role played by SQSTM1 in w09-induced apoptosis. In addition, in vivo administration of w09 effectively inhibited tumor growth of SGC-7901 xenografts. Hence, our findings not only suggested that activation of the EGFR-RAS-RAF1-MAP2K-MAPK1/3 signaling pathway may play a critical role in w09-induced autophagy and apoptosis, but also imply that induction of autophagic cancer cell death through activation of the EGFR pathway may be a potential therapeutic strategy for EGFR-disregulated gastric tumors.

Neferine augments therapeutic efficacy of cisplatin through ROS- mediated non-canonical autophagy in human lung adenocarcinoma (A549 cells)

Combination of dietary components with chemotherapy drugs is an emerging new strategy for cancer therapy to increase antitumor responses. Neferine, major bisbenzylisoquinoline alkaloid isolated from the seed embryo of Nelumbo nucifera (Lotus). In the present study, we investigated the efficacy of the combinatorial regimen of neferine and cisplatin compared to cisplatin high dose in human lung adenocarcinoma (A549) cells. Co-treatment with neferine enhanced cisplatin-induced autophagy in A549 cells was accompanied by Acidic vesicular accumulation (AVO), enhanced generation of reactive oxygen species (ROS) and depletion of intracellular glutathione (GSH), down regulation of PI3K/AKT/mTOR pathway, conversion of LC3B-I to LC3B-II. This enhanced autophagy developed via a non-canonical mechanism that did not require Beclin-1, PI3KCIII. In conclusion, these results suggest that neferine enhances cisplatin –induced autophagic cancer cell death through downregulation of PI3K/Akt/mTOR signaling pro-survival pathway and ROS- mediated Beclin-1 and PI3K CIII independent autophagy in human lung adenocarcinoma (A549 cells).

Berberine-induced autophagic cell death by elevating GRP78 levels in cancer cells.

Berberine, an isoquinoline alkaloid extracted from Coptidis Rhizoma, has been shown to induce cancer cell autophagic death. Glucose regulated protein 78 (GRP78) is associated with stress-induced autophagy. However, the related mechanisms between berberine-induced cancer cell autophagy and GRP78 remain to be elucidated. Here, we report that berberine can induce autophagic cancer cell death by elevating levels of GRP78. These results further demonstrated that berberine enhanced GRP78 by suppression of ubiquitination / proteasomal degradation of GRP78 and activation of ATF6. Moreover, fluorescence spectrum assay revealed that berberine could bind to GRP78 and form complexes. Finally, co-IP analysis showed that the ability of GRP78 to bind to VPS34 was increased with berberine treatment. Taken together, our results suggest that berberine induces autophagic cancer cell death via enhancing GRP78 levels and the ability of GRP78 to bind to VPS34.

Abstract 1151: IL-8, VEGF and IGF-1 play the important role in ARHI mediated-tumor dormancy in ovarian cancer

Abstract Ovarian cancer is the most lethal gynecologic malignancy. One of the most important factors contributing to poor outcomes is the ability of drug resistant ovarian cancer cells to remain dormant for years after treatment, only to grow progressively and kill their host. Judging from sites of recurrence, most dormant ovarian cancer cells are found on the surface of the peritoneal cavity, often in small, poorly vascularized collagenous scars observed at “second look” operations. Previously, we reported that more than 80% of persistent, dormant drug resistant ovarian cancers express ARHI and are undergoing autophagy. Autophagy can protect or kill tumor cells depending upon the context. Our group has found that autophagy and tumor dormancy can be regulated by an imprinted tumor suppressor gene, ARHI (DIRAS3), which is downregulated in 60% of ovarian cancers. Re-expression of ARHI in cell culture produces cell death within 72 hours, whereas re-expression of ARHI in xenografts produces cell growth arrest and tumor dormancy. When ARHI levels are reduced after 6 weeks of induction, xenografts grow promptly. Our current experiments were designed to determine whether survival of autophagic cells in vivo requires permissive levels of VEGF, IL-8 and IGF-1 in the tumor microenvironment. In cell culture, co-incubation of VEGF, IGF-1 and IL-8 with SKOv3-ARHI cells, increased AKT activity, reduced ARHI-mediated autophagy formation and decreased autophagic cell death. SKOv3-ARHI cells grown subcutaneously in nude mice exhibited higher levels of AKT activity and lower levels of autophagy than cells grown in cell culture. When cytoplasmic and nuclear fractions were obtained from xenografts, the nuclear fractions showed increased phospho-AKT, and decreased levels of a transcription factor EB (TFEB) and low mRNA expression of LC3, suggesting survival factors in tumor environment may stimulate AKT, downregulate expression of TFEB and inhibit formation of autophagy. Treatment with anti-VEGF, anti-IL-8 and anti-IGF-1 antibodies, individually and in combination, inhibited survival factors-mediated AKT activation and slowed the outgrowth of dormant autophagic ovarian cancer cells. Treatment of dormant xenografts with the same monoclonal antibodies against VEGF, IL-8 and IGFR not only delayed outgrowth when DOX is withdrawn, but cured a fraction of mice. Re-expression of ARHI reduced expression of IGF-1 receptor, but not VEGF and IL8 receptors. Treatment ovarian cancer cells with VEGF, IL-8 and IGF-1 reduced ARHI's ability to induce autophagy and to inhibit AKT. These preclinical studies support that the possibility that treatment with a combination of anti-VEGF, anti-IL8 and anti-IGF-1 antibodies could prevent outgrowth of dormant cells in patients with ovarian cancer. Furthermore, these results suggest that survival factors are required to limit autophagic death. Citation Format: Zhen Lu, Aaron Orozco, Weiqun Mao, Yan Wang, Margie Nicole Sutton, Hailing Yang, Douglas A. Levine, Robert C. Bast Jr.. IL-8, VEGF and IGF-1 play the important role in ARHI mediated-tumor dormancy in ovarian cancer. [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 1151.