Chemotherapy-induced Autophagy Research Articles

Autophagy Inhibition and Sensitization to Cisplatin in Esophageal Cancer Stem-Like Cells via All-trans Retinoic Acid-induced miR-30a.

Providing insights into the chemoresistance of esophageal squamous cell carcinoma (ESCC) and its dependence on chemotherapy-induced autophagy. Autophagy is induced during chemotherapy of cancer cells, promoting resistance to anti-cancer treatments. The objective of this study is to investigate the modulation of microRNA-30a (miR-30a), a known regulator of autophagy, in ESCC cells by all-trans retinoic acid (ATRA). Treatment involved ESCC cells KYSE-30 and TE8 with cis-dichloro-diamine platinum (CDDP), enriching CDDP-surviving cells (CDDP-SCs). qRT-PCR and dual luciferase reporter assay (DLRA) were employed to evaluate miR-30a expression and its interaction with Beclin-1 (BECN1) in both CDDP-SCs and those treated with ATRA. Chemotherapy using CDDP led to a significant decrease in miR-30a expression within ESCC cells. Increased autophagy levels were identified in cancer cells exhibiting stem cell-like properties, characterized by the overexpression of specific stem cell markers. These results suggest that the downregulation of miR-30a induced by CDDP treatment may represent a potential underlying mechanism for increased autophagic activity, as evidenced by the upregulation of autophagy-related proteins, such as BECN1 and an elevated LC3-II/LC3-I ratio. ATRA treatment elevated miR-30a expression and disrupted hallmark cancer stem cell (CSC) features in ESCC cells. Further investigations demonstrated that increased miR-30a expression led to a reduction in the expression of its target gene, BECN1, and attenuated BECN1-mediated autophagy. This resulted in an augmentation of CDDP-induced apoptosis in ESCC cells and a G2/M cell cycle arrest. CDDP chemotherapy reduced miR-30a, promoting ESCC cell resistance through autophagy and CSC-like features, a process that may be modulated by ATRA.

The Two-Faced Role of Autophagy in Endometrial Cancer.

Autophagy, meaning “self-eating,” is a cellular catabolic process that involves lysosomal degradation of cytoplasmic materials. Autophagy contributes to both quality control and energy supply of cells, which are associated with tumorigenesis and tumor development, respectively. Endometrial cancer (EC) is the most common gynecologic cancer, and its incidence is increasing. Although autophagy plays crucial roles in several types of cancer, such as pancreatic ductal adenocarcinoma, its role in EC has not been clearly demonstrated. Activation of the PI3K/AKT/mTOR pathway, which functions to suppress autophagy, is an initial step in type 1 endometrial carcinogenesis, whereas a loss-of-function mutation of TP53, which augments autophagy via p16 induction, is the main cause of type 2 endometrial carcinogenesis. Mutations in autophagy-related genes, including ATG4C, RB1CC1/FIP200, and ULK4, have been reported in EC; thus, an aberrant autophagy mechanism may be involved in endometrial carcinogenesis. Furthermore, the biguanide diabetes drug metformin, treatment with which enhances autophagy via AMPK-mediated mTOR inactivation, has been reported to reduce the risk of EC. These findings suggest that autophagy negatively regulates endometrial carcinogenesis, and autophagy inducers may be useful for chemoprevention of EC. In contrast, autophagy appears to promote EC once it is established. Consistent with this, treatment with chloroquine, an autophagy inhibitor, is reported to attenuate EC cell proliferation. Moreover, chemotherapy-induced autophagy triggers chemoresistance in EC cells. As autophagy has a tumor-promoting function, the combination of chemotherapy and autophagy inhibitors such as chloroquine could be a potent therapeutic option for patients with EC. In conclusion, autophagy plays a dual role in the prevention and treatment of EC. Therefore, targeting autophagy to prevent and treat EC requires diametrically opposed strategies.

Biomimetic nanoparticles blocking autophagy for enhanced chemotherapy and metastasis inhibition via reversing focal adhesion disassembly

BackgroundAutophagy is a conserved catabolic process, which plays an important role in regulating tumor cell motility and degrading protein aggregates. Chemotherapy-induced autophagy may lead to tumor distant metastasis and even chemo-insensitivity in the therapy of hepatocellular carcinoma (HCC). Therefore, a vast majority of HCC cases do not produce a significant response to monotherapy with autophagy inhibitors.ResultsIn this work, we developed a biomimetic nanoformulation (TH-NP) co-encapsulating Oxaliplatin (OXA)/hydroxychloroquine (HCQ, an autophagy inhibitor) to execute targeted autophagy inhibition, reduce tumor cell migration and invasion in vitro and attenuate metastasis in vivo. The tumor cell-specific ligand TRAIL was bioengineered to be stably expressed on HUVECs and the resultant membrane vesicles were wrapped on OXA/HCQ-loaded PLGA nanocores. Especially, TH-NPs could significantly improve OXA and HCQ effective concentration by approximately 21 and 13 times in tumor tissues compared to the free mixture of HCQ/OXA. Moreover, the tumor-targeting TH-NPs released HCQ alkalized the acidic lysosomes and inhibited the fusion of autophagosomes and lysosomes, leading to effective blockade of autophagic flux. In short, the system largely improved chemotherapeutic performance of OXA on subcutaneous and orthotopic HCC mice models. Importantly, TH-NPs also exhibited the most effective inhibition of tumor metastasis in orthotopic HCCLM3 models, and in the HepG2, Huh-7 or HCCLM3 metastatic mice models. Finally, we illustrated the enhanced metastasis inhibition was attributed to the blockade or reverse of the autophagy-mediated degradation of focal adhesions (FAs) including E-cadherin and paxillin.ConclusionsTH-NPs can perform an enhanced chemotherapy and antimetastatic effect, and may represent a promising strategy for HCC therapy in clinics.Graphical

Molecular imaging of the kinetics of hyperactivated ERK1/2-mediated autophagy during acquirement of chemoresistance

Alterations in key kinases and signaling pathways can fine-tune autophagic flux to promote the development of chemoresistance. Despite empirical evidences of strong association between enhanced autophagic flux with acquired chemoresistance, it is still not understood whether an ongoing autophagic flux is required for both initiation, as well as maintenance of chemoresistance, or is sufficient for one of the either steps. Utilizing indigenously developed cisplatin–paclitaxel-resistant models of ovarian cancer cells, we report an intriguing oscillation in chemotherapy-induced autophagic flux across stages of resistance, which was found to be specifically elevated at the early stages or onset of chemoresistance. Conversely, the sensitive cells and cells at late stages of resistance showed stalled and reduced autophagic flux. This increased flux at early stages of resistance was found to be dictated by a hyperactive ERK1/2 signaling, which when inhibited either pharmacologically (U0126/Trametinib) or genetically, reduced p62 degradation, number of LC3+veLAMP1+ve puncta, autophagolysosome formation, and led to chemo-sensitization and apoptosis. Inhibition of ERK1/2 activation also altered the level of UVRAG and Rab7, the two key proteins involved in autophagosome–lysosome fusion. Noninvasive imaging of autophagic flux using a novel autophagy sensor (mtFL-p62 fusion reporter) showed that combinatorial treatment of platinum–taxol along with Trametinib/chloroquine blocked autophagic flux in live cells and tumor xenografts. Interestingly, Trametinib was found to be equally effective in blocking autophagic flux as chloroquine both in live cells and tumor xenografts. Combinatorial treatment of Trametinib and platinum–taxol significantly reduced tumor growth. This is probably the first report of real-time monitoring of chemotherapy-induced autophagy kinetics through noninvasive bioluminescence imaging in preclinical mouse model. Altogether our data suggest that an activated ERK1/2 supports proper completion of autophagic flux at the onset of chemoresistance to endure initial chemotherapeutic insult and foster the development of a highly chemoresistant phenotype, where autophagy becomes dispensable.

Abstract A03: Autophagy and HSP27: A potential link to define autophagy fate in osteosarcoma

Abstract Survival in osteosarcoma (OS) has not improved in over a decade, thus the need for new therapeutic strategies. Autophagy, a catabolic process used by cells to survive under stress, has been implicated in resistance to chemotherapy. We previously demonstrated aerosol gemcitabine (GCB) efficacy against OS lung metastasis and showed GCB to induce autophagy through the Akt/mTOR signaling pathway. Whether autophagy contributes to tumor response or resistance to GCB has been the focus of our studies. What determines whether chemotherapy-induced autophagy will lead to survival or death is unknown. We found phosphorylated heat shock protein 27 (pHSP27) to potentially define the outcome of chemotherapy-induced autophagy in OS. Small heat shock proteins, specifically HSP27, are upregulated in many cancers, including OS, and associated with treatment resistance. The purpose of this study is to investigate if induction of pHSP27 predicts the role of chemotherapy-induced autophagy in OS lung metastasis. We hypothesize that when pHSP27 is induced, blocking autophagy will lead to increased sensitivity to GCB. Furthermore, inhibition of pHSP27 may revert the effect of GCB-induced autophagy in OS cells by decreasing sensitivity to GCB. We showed in the LM7 and CCH-OS-D human metastatic OS cells in vitro, that GCB induces autophagy as determined by the conversion of microtubule-associated light chain 3 one to two (LC3I/LC3II), increase in Beclin 1, and decrease in p62 protein expression. Sensitivity of LM7 cells to GCB was enhanced after autophagy inhibition by hydroxychloroquine (HCQ), a pharmacologic inhibitor and shRNA targeting Beclin 1, suggesting autophagy as a prosurvival mechanism whereas in CCH-OS-D cells inhibition of autophagy decreased cell sensitivity to GCB, concluding here that induction of autophagy leads to cell death. We demonstrated that pHSP27 is significantly higher in GCB-treated LM7 cells as compared to CCH-OS-D cells. We also showed that inhibition of HSP27 by shRNA targeting HSP27 has no effect on GCB-induced autophagy in LM7 cells. Treatment of shHSP27LM7 with GCB resulted in an increase in LC3I/LC3II conversion and a decrease in p62. We further revealed this in vivo by conducting a pilot study in which shHSP27LM7 cells were injected i.v. into nude mice. 6 weeks later, lung metastases were confirmed. Animals were divided into two groups: untreated and aerosol GCB treated (1mg/mL three times a week). Mice were sacrificed after 2 weeks of therapy. Transmitted electron microscopy demonstrated an increase in the number of autophagosomes in lung metastasis from mice treated with GCB, confirming induction of autophagy in the absence of HSP27. Lastly, in order to demonstrate that inhibition of HSP27 will change GCB-induced autophagy response in LM7 cells, we treated shHSP27LM7 cells with the combination GCB+HCQ and showed increased viability compared to the LM7 GipZ control cells, confirming preliminarily our hypothesis that induction of pHSP27 will determine chemotherapy-induced autophagy response in OS. Citation Format: Grace Nehme, Kumar Felix, Andrew Wahba, Diana M. Fandino, Nancy Gordon. Autophagy and HSP27: A potential link to define autophagy fate in osteosarcoma [abstract]. In: Proceedings of the AACR Special Conference on Targeting PI3K/mTOR Signaling; 2018 Nov 30-Dec 8; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(10_Suppl):Abstract nr A03.

LINC01572 Regulates Cisplatin Resistance in Gastric Cancer Cells by Mediating miR-497-5p.

BackgroundChemotherapy resistance has long been recognized as a major obstacle to cancer treatment. Therefore, elucidating the underlying mechanisms of chemotherapy resistance is conducive to developing new strategies to improve patients’ response to chemotherapy drugs.Materials and MethodsReal-time quantitative PCR (QPCR) was applied to measure the expression levels of lncRNAs. LINC01572 was down-regulated or up-regulated in GC cells transfected with either LINC01572 shRNA or overexpression vectors. In vitro and in vivo experiments were conducted to investigate the role of LINC01572 in autophagy-related chemotherapy resistance.ResultsCompared with the parental cells, drug-resistant GC cells had a higher level of LINC01572. Silencing of LINC01572 inhibited chemotherapy-induced autophagy, while its knockout sensitized GC cells against chemotherapy drugs. As a competitive endogenous RNA of miR-497-5p, LINC01572 weakened the inhibitory effect of miR-497-5p on ATG14, leading to chemically induced autophagy and chemotherapy resistance in GC cells.ConclusionA new mechanism of GC autophagy-related chemotherapy resistance regulated by lncRNA was explored in this study, providing a new perspective for understanding chemotherapy resistance.

SIRT6-PARP1 is involved in HMGB1 polyADP-ribosylation and acetylation and promotes chemotherapy-induced autophagy in leukemia

ABSTRACT High mobility group box protein 1 (HMGB1) is an evolutionarily conserved non-histone chromatin-binding protein. In a previous study, we showed that treating leukemic cells with chemotherapeutic drugs leads to the translocation of HMGB1, which is involved in autophagy and ultimately promotes chemoresistance in leukemia. However, the underlying translocation mechanism of HMGB1 in chemotherapy-induced autophagy remains unclear. In this study, we showed that knockdown of SIRT6 or PARP1 gene expression significantly inhibited HMGB1 cytoplasmic translocation and autophagy. Meanwhile, we found that SIRT6, an important upstream protein of PARP1, associated with PARP1, leading to the stimulation of polyADP-ribose polymerase activity. We further demonstrated that SIRT6 and PARP1 activation were required for chemotherapy-induced ADP-ribosylation of HMGB1 in leukemic cells and then influenced the acetylation of HMGB1, finally promoting the autophagy of leukemic cells mediated by HMGB1 translocation. These findings provide new insights into the mechanism of chemotherapeutic drug resistance. Targeting the HMGB1 translocation may overcome autophagy-related chemoresistance in leukemia.

The Switch between Protective and Nonprotective Autophagy; Implications for Autophagy Inhibition as a Therapeutic Strategy in Cancer

Autophagy, a process of cellular self-degradation and cell survival whereby the cell generates energy and metabolic intermediates under conditions of stress (i.e., nutrient deprivation), is also commonly induced in tumor cells in response to chemotherapy and radiation. While chemotherapy-induced autophagy and radiation-induced autophagy are generally considered to have cytoprotective functions, thereby reducing tumor cell sensitivity (and potentially conferring resistance) to various treatment modalities, autophagy can also be nonprotective; furthermore, the nature of the autophagy can be altered via the “autophagic switch” depending on such factors as the p53 status of the tumor cells. Defective or compromised autophagy has also been associated with neurodegenerative diseases, raising concerns as to the impact of autophagy inhibition on normal tissue function. Furthermore, the impact of autophagy inhibition on the immune system response to therapy as well as the influence of autophagy inhibition in combination with chemotherapy or radiation on critical tissue sites such as the bone marrow remain uncertain. These are factors requiring serious consideration within the context of current clinical efforts to exploit autophagy inhibition as a therapeutic strategy in cancer.

Poly (ADP-ribosylation) of HMGB1 facilitates its acetylation and promotes HMGB1 translocation-associated chemotherapy-induced autophagy in leukaemia cells.

Acute lymphoblastic leukaemia (ALL) is one of the most common and curable types of cancer in paediatric patients. However, chemotherapeutic resistance is a difficult but common obstacle when treating leukaemia in the clinical setting. Studies have demonstrated that drug resistance is partly attributable to autophagy induced by multiple chemotherapeutic agents. As an evolutionarily conserved non-histone chromatin-binding protein, high mobility group box protein 1 (HMGB1) is considered to be an important factor in autophagy, and regulates autophagy at multiple levels via different subcellular localisations. In the present study, it was revealed that chemotherapeutic drugs induced autophagy in leukaemia cells and that translocation of HMGB1 from the nucleus to the cytoplasm is an important molecular event in this process. It was further demonstrated that poly (ADP-ribosylation) of HMGB1 facilitates its acetylation, thereby inducing HMGB1 translocation and ultimately promoting chemotherapy-induced autophagy in leukaemic cells. Targeted HMGB1 translocation may overcome chemotherapy-induced autophagy in leukaemia.

Abstract 1330: Role of epigenetic remodeling in sensitizing triple-negative breast cancer cells to treatment through enhanced chemotherapy-induced autophagy

Abstract Dysregulation of the epigenome is implicated in initiation and progression of variety of cancers and their acquired resistance to chemotherapy. As such targeting epigenetic regulators has the potential to modulate cancer cell biology and their sensitivity to chemotherapy and/or radiation. Our studies demonstrates that silencing of the epigenetic regulator Nucleosome Remodeling Factor (NURF) sensitizes breast tumor cells to chemotherapy and enhances the anti-tumor immune response. A screen of a variety of chemotherapeutic agents shows that NURF KD cells are selectively sensitized to Topo II inhibitors, which includes doxorubicin. NURF KD increases DNA damage (gamma H2AX staining) and autophagy (acridine orange staining) in breast tumor cells exposed to doxorubicin (Dox) and enhances growth inhibition as well as suppressing the ability of the cells to recover proliferative capacity. Increased autophagy as well as breast tumor sensitization to doxorubicin were observed using a small molecule inhibitor of NURF, suggesting that NURF can also be targeted pharmacologically. Our studies suggest that enhanced autophagy may be a primary contributor to chemo sensitivity in NURF KD cells. Studies are currently underway to confirm the role of autophagy in chemosensitization in- vitro and enhanced immune response in-vivo using autophagy deficient cells (through ATG silencing). Doxorubicin produces profound DNA damage and promotes immunogenic cell death enhanced by NURF KD. Increased cell autonomous antitumor effects by doxorubicin in concert with increased cell non-autonomous antigenicity could help to achieve tumor regression, reduce metastasis, and possibly promote long term remission in breast cancer. Citation Format: Liliya Tyutyunyk, Joseph Landry, David Gewirtz, Nga Dao. Role of epigenetic remodeling in sensitizing triple-negative breast cancer cells to treatment through enhanced chemotherapy-induced autophagy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1330.

Temozolomide Enhances Triple-Negative Breast Cancer Virotherapy In Vitro.

Triple-negative breast cancer (TNBC) is one of the most aggressive types of cancer, and treatment is limited to chemotherapy and radiation. Oncolytic virotherapy may be a promising approach to treat TNBC. However, oncolytic adenovirus (OAd)-based mono-therapeutic clinical trials have resulted in modest outcomes. The OAd potency could be increased by chemotherapy-induced autophagy, an intracellular degradation system that delivers cytoplasmic constituents to the lysosome. In this study, the ability of alkylating agent temozolomide (TMZ)-induced autophagy to increase OAd replication and oncolysis in TNBC cells was evaluated. Human TNBC MDA-MB-231 and HCC1937 cells and mouse 4T1 cells were infected with an OAd expressing the red fluorescent protein mCherry on the virus capsid (OAdmCherry) alone or in combination with TMZ. TNBC cells treated with OAdmCherry/TMZ displayed greater mCherry and adenovirus (Ad) early region 1A (E1A) expression and enhanced cancer-cell killing compared to OAdmCherry or TMZ alone. The combined therapy-mediated cell death was associated with virus replication and accumulation of the autophagy marker light chain 3 (LC3)-II. Overall, this study provides experimental evidence of TMZ’s ability to increase oncolytic virotherapy in both human and murine TNBC cells.

Autophagy-deficient breast cancer shows early escape from dormancy and recurrence following chemotherapy

Abstract Breast cancer patients who respond to cancer therapies often end up with a distant recurrence of the disease. It is not clear why people with the same type of breast cancer respond to treatments differently, some escape from dormancy and relapse earlier than others. In addition, some tumor clones respond to immunotherapy while others do not. Here, we investigated the role of autophagy in expediting or delaying recurrence of neu overexpressing mammary carcinoma (MMC) following adriamycin (ADR) chemotherapy, and in affecting response to immunotherapy. We took two strategies, including a transient blockade of autophagy with chloroquine (CQ) which transiently blocks fusion of autophagosome and lysosome, and a stable knockdown of autophagy, which inhibits the formation of autophagosome in MMC. We found that while CQ prolonged tumor dormancy, a stable knockdown of autophagy resulted in early escape from dormancy and recurrence. Interestingly, MMC with a stable knockdown of autophagy contained an increased frequency of ADR-induced polyploidy cells which rendered MMC resistant to immunotherapy. On the other hand, a transient blockade of autophagy did not affect the sensitivity of MMC to immunotherapy. Our observations suggest that while chemotherapy-induced autophagy may facilitate tumor relapse, cell intrinsic autophagy delays tumor relapse by inhibiting the formation of polyploidy tumor cells. Our findings are consistent with other reports showing that autophagy-deficient cells contain polyploid nuclei because of increased levels of reactive oxygen species. Our results also suggest that responsiveness of breast cancer patients to neoadjuvant therapies might be determined by tumor intrinsic autophagy.

Autophagy-deficient breast cancer shows early tumor recurrence and escape from dormancy.

Breast cancer patients who initially respond to cancer therapies often succumb to distant recurrence of the disease. It is not clear why people with the same type of breast cancer respond to treatments differently; some escape from dormancy and relapse earlier than others. In addition, some tumor clones respond to immunotherapy while others do not. We investigated how autophagy plays a role in accelerating or delaying recurrence of neu-overexpressing mouse mammary carcinoma (MMC) following adriamycin (ADR) treatment, and in affecting response to immunotherapy. We explored two strategies: 1) transient blockade of autophagy with chloroquine (CQ), which blocks fusion of autophagosomes and lysosomes during ADR treatment, and 2) permanent inhibition of autophagy by a stable knockdown of ATG5 (ATG5KD), which inhibits the formation of autophagosomes in MMC during and after ADR treatment. We found that while CQ prolonged tumor dormancy, but that stable knockdown of autophagy resulted in early escape from dormancy and recurrence. Interestingly, ATG5KD MMC contained an increased frequency of ADR-induced polyploid-like cells and rendered MMC resistant to immunotherapy. On the other hand, a transient blockade of autophagy did not affect the sensitivity of MMC to immunotherapy. Our observations suggest that while chemotherapy-induced autophagy may facilitate tumor relapse, cell-intrinsic autophagy delays tumor relapse, in part, by inhibiting the formation of polyploid-like tumor dormancy.

MiRNA-409-3p enhances cisplatin-sensitivity of ovarian cancer cells by blocking the autophagy mediated by Fip200.

Background: Efficacy of chemotherapy for ovarian cancer (OC) is usually affected by various factors,especially the chemoresistance of ovarian cancer cells. Downregulation of miR-409-3p has been found in a variety of tumors. However, the role of miR-409-3p in chemo-resistant OC cells still remains unknown. The aim of this study was to investigate the effect of miR-409-3p on cisplatin-resistant OC cells and to elucidate the mechanism by which enhances cisplatin-sensitivity of OC cells. Methods: Expression of miR-409-3p in OC cells was assessed by qRT-PCR. MTS and clone formation assays were used to validate cell proliferation. Flow cytometry was performed for apoptosis analysis. Western blot assay was used to assess the alterations of signaling pathway related proteins. BALB/c nude mouse xenograft model was used to evaluate the role of miR-409-3p in cisplatin-resistant OC cells in vivo. Results: We found that miR-409-3p was apparently downregulated in the OC cells compared with normal ovarian cells. Results also showed that compared with the cisplatinsensitive cells, in cisplatin-resistant cells, miR-409-3p was decreased with an obvious increasing autophagic activity. In addition, based on the bioinformatics analysis, miR-409-3p was supposed to bind with Fip200. Our results demonstrated that in miR-409-3p overexpression cells, significant decreases were seen in protein expression of Fip200 and autophagic activity, which might be caused by conjugation between overexpressed miR-409-3p and 3'-UTR in Fip200 mRNA. Moreover, under the miR-409-3p overexpression, we found that cisplatin-sensitive and cisplatin-resistant ovarian cancer cells were more sensitive to the chemotherapeutics, which could be reversed by Fip2000. Further, we found that chemosensitivity in tumor cells was augmented by miR-409-3p overexpression, and Fip200 acted as a key link in interrupting the chemotherapy-induced autophagy. Conclusions: Results mentioned above revealed that miR-409-3p can ameliorate cisplatin-sensitivity of ovarian cancer cells through blocking the autophagy mediated by Fip200.

Phosphorylated heat shock protein 27 as a potential biomarker to predict the role of chemotherapy-induced autophagy in osteosarcoma response to therapy

Autophagy is a catabolic process involved in cellular homeostasis. Autophagy is increased above homeostatic levels by chemotherapy, and this can either promote or inhibit tumor growth. We previously demonstrated that aerosol gemcitabine (GCB) has a therapeutic effect against osteosarcoma (OS) lung metastases. However, some tumor cells failed to respond to the treatment and persisted as isolated lung metastasis. Here, we examined the mechanisms underlying the dual role of chemotherapy-induced autophagy in OS and sought to identify biomarkers to predict OS response to treatment. In this study, we demonstrate that treatment of various OS cells with GCB induced autophagy. We also showed that GCB reduces the phosphorylation of AKT, mTOR and p70S6K and that GCB-induced autophagy in OS can lead to either cell survival or cell death. Blocking autophagy enhanced the sensitivity of LM7 OS cells and decreased the sensitivity of CCH-OS-D and K7M3 OS cells to GCB. Using a kinase array, we also demonstrated that differences in the phosphorylated heat shock protein 27 (p-HSP27) expression in the various OS cell lines after treatment with GCB, correlates to whether chemotherapy-induced autophagy will lead to increase or decrease OS cells sensitivity to therapy. Increased p-HSP27 was associated with increased sensitivity to anticancer drug treatment when autophagy is inhibited. The results of this study reveal a dual role of autophagy in OS cells sensitivity to chemotherapy and suggest that p-HSP27 could represent a predictive biomarker of whether combination therapy with autophagy modulators and chemotherapeutic drugs will be beneficial for OS patients.

Abstract 3314: Synergistic effects of chemotherapy-induced autophagy and epigenetic remodeling

Abstract Cytotoxic chemotherapy has been shown to enhance tumor cell immunogenicity by promoting the secretion of damage-associated molecular pattern (DAMP) molecules which, in turn, stimulates the immune response. These effects can be further stimulated by combining chemotherapy with an immunotherapy designed to enhance effector cell (CD8 T cell or NK cell) anti-tumor activity. Our studies demonstrate that knockdown of the epigenetic regulator NURF increases DNA damage (gamma H2AX staining) and autophagy (acridine orange staining) in breast tumor cells exposed to doxorubicin and enhances growth inhibition as well as suppressing the capacity of the cells to recover and proliferate. Similar increases in autophagy were observed using a small molecule inhibitor of NURF, suggesting that NURF can be targeted therapeutically. Sensitization was not observed with ionizing radiation or cisplatin. Studies are in progress to assess the nature of autophagy in the NURF KD and NURF WT cells, distinguishing between the cytoprotective, cytotoxic, cytostatic and nonprotective forms. How NURF regulates doxorubicin induced DNA damage is being investigated by mapping sites of damage genome wide. It is anticipated that enhanced cell autonomous tumor cell sensitivity in concert with improvements in tumor cell antigenicity (cell non-autonomous sensitization) achieved by NURF depletion could improve anti-tumor immunogenicity, achieve tumor regression, reduce metastasis, and possibly achieve long term remission in breast cancer. Citation Format: Liliya Tyutyunyk, Joseph Landry, Tareq Saleh, David Gewirtz. Synergistic effects of chemotherapy-induced autophagy and epigenetic remodeling [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 3314. doi:10.1158/1538-7445.AM2017-3314

MiR-34a Promotes Apoptosis and Inhibits Autophagy by Targeting HMGB1 in Acute Myeloid Leukemia Cells

MiR-34a is identified as a tumor suppressor gene and involved in acute myeloid leukemia (AML) development. However, the regulatory mechanism of miR-34a in AML is unclear. The expression of miR-34a and HMGB1 in HL-60, THP-1 and HS-5 cells were detected by qRT-PCR and western blot. Lipofectamine 2000 was used to transfect with miR-34a mimics, miR-34a inhibitor, si-HMGB1, pcDNA 3.1-HMGB1, and corresponding controls. The apoptosis and autophagy of transfected AML cells were assessed by flow cytometry and western blot, respectively. Bioinformatics software and dual luciferase reporter assay were applied to predict and verify the target of miR-34a. The effects of miR-34a mimics or si-HMGB1 on chemotherapy-induced autophagy were further explored in HL-60 cells treated with all-trans retinoic acid (ATRA) along with lysosomal protease inhibitors E64d and pepstatin A. MiR-34a was lower expressed and HMGB1 mRNA and proteins were both higher expressed in HL-60 and THP-1 cells compared with that in HS-5 cells. Higher expression levels of MiR-34 and lower expression levels of HMGB1 both significantly promoted apoptosis and inhibited autophagy in HL-60 and THP-1 cells. Dual luciferase reporter system confirmed that HMGB1 was a potential target of miR-34a. Moreover, overexpression of HMGB1 dramatically reversed the promotion of apoptosis and inhibition of autophagy mediated by higher expression level of miR-34a. Higher expression level of miR-34a and lower expression level of HMGB1 both inhibited chemotherapy-induced autophagy by stimulating the LC3 conversion. MiR-34a promoted cell apoptosis and inhibited autophagy by targeting HMGB1. Therefore, miR-34a may be a potential promising molecular target for AML therapy.

The Complex Link between Apoptosis and Autophagy: a Promising New Role for RB.

Physiological processes, as autophagy, proliferation and apoptosis are affected during carcinogenesis. Restoring cellular sensitivity to apoptotic stimuli, such as the antineoplastic cocktails, has been explored as a strategy to eliminate cancer cells. Autophagy, a physiological process of recycling organelles and macromolecules can be deviated from homeostasis to support cancer cells survival, proliferation, escape from apoptosis, and therapy resistance. The relationship between autophagy and apoptosis is complex and many stimuli can induce both processes. Most chemotherapeutic agents induce autophagy and it is not clear whether and how this chemotherapy-induced autophagy might contribute to resistance to apoptosis. Here, we review current strategies to sensitize cancer cells by interfering with autophagy. Moreover, we discuss a new link between autophagy and apoptosis: the tumor suppressor retinoblastoma protein (RB). Inactivation of RB is one of the earliest and more frequent hallmarks of cancer transformation, known to control cell cycle progression and apoptosis. Therefore, understanding RB functions in controlling cell fate is essential for an effective translation of RB status in cancer samples to the clinical outcome.

Late-stage inhibition of autophagy enhances calreticulin surface exposure.

Calreticulin (CRT) exposure on the cell surface is essential for inducing immunogenic cell death by chemotherapy. Recent studies have shown conflicting effects of chemotherapy-induced autophagy on CRT exposure in cancer cells. Our data revealed that surface-exposed CRT (Ecto-CRT) emission was attenuated by inhibition of autophagy at early stages; however, inhibition of autophagy at late stages resulted in increased Ecto-CRT. Furthermore, neither autophagy activation nor endoplasmic reticulum (ER) stress induction alone was sufficient for CRT surface exposure. Moreover, chemotherapeutic agents that only activated autophagy without inducing ER stress could not increase Ecto-CRT; therefore, combined use of an autophagy activator and ER stress inducer could effectively promote CRT translocation to the plasma membrane. Together, our results highlight the potential of the combined use of ER stress inducers and autophagy late-stage inhibitors to reestablish and strengthen both the CRT exposure and immunogenicity of chemotherapeutic agents induced death cells.

MiR-140-5p attenuates chemotherapeutic drug-induced cell death by regulating autophagy through inositol 1,4,5-trisphosphate kinase 2 (IP3k2) in human osteosarcoma cells.

Acquisition of drug-resistant phenotypes is often associated with chemotherapy in osteosarcoma. A number of studies have demonstrated a critical role for autophagy in osteosarcoma development, therapy and drug resistance. However, the molecular mechanisms underlying the autophagy-mediated chemotherapy resistance of osteosarcoma cells remain largely unknown. In the present study, we determined the autophagy and microRNA-140 (miR-140-5p, miRBase ID: MIMAT0000431) expression induced by chemotherapeutic drugs in osteosarcoma cells. Then we determined the promotory role of miR-140-5p to the chemotherapy-induced autophagy. Our results demonstrated that miR-140-5p expression was highly induced during chemotherapy of osteosarcoma cells, and this was accompanied by up-regulated autophagy. The increased miR-140-5p expression levels up-regulated anticancer drug-induced autophagy in osteosarcoma cells and ameliorated the anticancer drug-induced cell proliferation and viability decrease. Importantly, miR-140-5p regulates this context-specific autophagy through its target, inositol 1,4,5-trisphosphate kinase 2 (IP3k2). Therefore, the results of the present study demonstrated that miR-140-5p mediated drug-resistance in osteosarcoma cells by inducing autophagy. The present study provides evidence of miRNA regulation of autophagy through modulation of IP3 signalling. The present study recognized a novel mechanism of chemoresistance in osteosarcoma cancers.