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Autophagy-independent induction of LC3B through oxidative stress reveals its non-canonical role in anoikis of ovarian cancer cells.

ABSTRACT: Cancer cells display abnormal redox metabolism. Autophagy, anoikis and reactive oxygen species (ROS) play a regulatory role during metastasis. LC3 is a well-known essential molecule for autophagy. Therefore, we wanted to explore the molecular interplay between autophagy, anoikis, and ROS in relation to LC3B. We observed enhanced LC3B level along with increased expression of p62 and modulation of other autophagy-related molecules (Atg 3, 5, 7, 12, 16L1 and Beclin1) by inducing oxidative-stress in ovarian cancer cells using a ROS-producing pro-oxidant molecule. Surprisingly, enhanced LC3B was unable to induce autophagosome formation rather promoted anoikis. ROS-induced inhibition of autophagosome-formation is possibly due to the instability of autophagy initiator, ULK1 complex. Moreover, such upregulation of LC3B via ROS enhanced several apoptotic molecules. Silencing LC3B reduced these apoptotic molecules and increased when overexpressed, suggesting its role in apoptosis. Furthermore, LC3B-dependent apoptosis was decreased by inhibiting ROS, indicating a possible link between ROS, LC3B, and apoptosis. Additionally, ROS-induced enhanced LC3B promoted detachment-induced cell death (anoikis). This was further reflected by reduced cell adhesion molecules (integrin-?3 and focal adhesion kinase) and mesenchymal markers (snail and slug). Our in vitro experimental data was further confirmed in primary tumors developed in syngeneic mice, which also showed ROS-mediated LC3B enhancement along with reduced autophagosomes, integrin-?3 and focal adhesion kinase ultimately leading to the decreased tumor mass. Additionally, primary cells from high-grade serous carcinoma patient's ascites exhibited LC3B enhancement and autophagy inhibition through ROS which provided a clinical relevance of our study. Taken together, this is the first evidence for a non-canonical role of LC3B in promoting anoikis in contrast to autophagy and may, therefore, consider as a potential therapeutic target molecule in ovarian cancer. Taken together, autophagy-inhibition may be an alternative approach to induce apoptosis/anoikis in cancer.

SUBMITTER: Satyavarapu EM

PROVIDER: S-EPMC6141567 | biostudies-literature | 2018 Sep

REPOSITORIES: biostudies-literature

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Autophagy-independent induction of LC3B through oxidative stress reveals its non-canonical role in anoikis of ovarian cancer cells.

Satyavarapu Eswara Murali EM Das Ranjita R Mandal Chandan C Mukhopadhyay Asima A Mandal Chitra C

Cell death & disease 20180917 10

Cancer cells display abnormal redox metabolism. Autophagy, anoikis and reactive oxygen species (ROS) play a regulatory role during metastasis. LC3 is a well-known essential molecule for autophagy. Therefore, we wanted to explore the molecular interplay between autophagy, anoikis, and ROS in relation to LC3B. We observed enhanced LC3B level along with increased expression of p62 and modulation of other autophagy-related molecules (Atg 3, 5, 7, 12, 16L1 and Beclin1) by inducing oxidative-stress in ...[more]

PMID: 30224639

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Project description:Peritoneal carcinomatosis and peritoneal sarcomatosis is a potential complication of nearly all solid tumors and results in profoundly increased morbidity and mortality. Despite the ubiquity of peritoneal carcinomatosis/peritoneal sarcomatosis, there are no clinically relevant targeted therapies for either its treatment or prevention. To identify potential therapies, we developed in vitro models of peritoneal carcinomatosis/peritoneal sarcomatosis using tumor cell lines and patient-derived spheroids (PDS) that recapitulate anoikis resistance and spheroid proliferation across multiple cancer types. Epithelial- and mesenchymal-derived cancer cell lines (YOU, PANC1, HEYA8, CHLA10, and TC71) were used to generate spheroids and establish growth characteristics. Differential gene expression analyses of these spheroids to matched adherent cells revealed a consensus spheroid signature. This spheroid signature discriminates primary tumor specimens from tumor cells found in ascites of ovarian cancer patients and in our PDS models. Key in this gene expression signature is BNIP3 and BNIP3L, known regulators of autophagy and apoptosis. Elevated BNIP3 mRNA expression is associated with poor survival in ovarian cancer patients and elevated BNIP3 protein, as measured by IHC, and is also associated with higher grade tumors and shorter survival. Pharmacologic induction of autophagy with rapamycin significantly increased spheroid formation and survival while decreasing the induction of apoptosis. In contrast, the autophagy inhibitor hydroxychloroquine abrogated spheroid formation with a clear increase in apoptosis. Modulation of BNIP3 and the critical autophagy gene Beclin-1 (BECN1) also caused a significant decrease in spheroid formation. Combined, these data demonstrate how modulation of BNIP3-related autophagy, in PDS and in vitro spheroid models, alters the survival and morphology of spheroids. IMPLICATIONS:Development of BNIP3/BNIP3L-targeting agents or autophagy-targeting agents may reduce morbidity and mortality associated with peritoneal carcinomatosis and sarcomatosis. Mol Cancer Res; 15(1); 26-34. ©2016 AACR.

Project description:BackgroundThe contribution of native or modified oligodendroglia-derived extracellular vesicles (OL-EVs) in controlling chronic inflammation is poorly understood. In activated microglia, OL-EVs contribute to the removal of cytotoxic proteins following a proteotoxic stress. Intracellular small heat shock protein B8 (HSPB8) sustain this function by facilitating autophagy and protecting cells against oxidative stress mediated cell death. Therefore, secretion of HSPB8 in OL-EVs could be beneficial for neurons during chronic inflammation. However, how secreted HSPB8 contribute to cellular proteostasis remains to be elucidated.MethodsWe produced oligodendroglia-derived EVs, either native (OL-EVs) or HSPB8 modified (OL-HSPB8-EVs), to investigate their effects in controlling chronic inflammation and cellular homeostasis. We analyzed the impact of both EV subsets on either a resting or activated microglial cell line and on primary mixed neural cell culture cells. Cells were activated by stimulating with either tumor necrosis factor-alpha and interleukin 1-beta or with phorbol-12-myristate-13-acetate.ResultsWe show that OL-EVs and modified OL-HSPB8-EVs are internalized by C20 microglia and by primary mixed neural cells. The cellular uptake of OL-HSPB8-EVs increases the endogenous HSPB8 mRNA expression. Consistently, our results revealed that both EV subsets maintained cellular homeostasis during chronic inflammation with an increase in the formation of autophagic vesicles. Both EV subsets conveyed LC3B-II and BAG3 autophagy markers with an enhanced effect observed for OL-HSPB8-EVs. Moreover, stimulation with either native or modified OL-HSPB8-EVs showed a significant reduction in ubiquitinated protein, reactive oxygen species and mitochondrial depolarization, with OL-HSPB8-EVs exhibiting a more protective effect. Both EV subsets did not induce cell death in the C20 microglia cell line or the primary mixed neural cultures.ConclusionWe demonstrate that the functions of oligodendroglia secreted EVs enriched with HSPB8 have a supportive role, comparable to the native OL-EVs. Further development of engineered oligodendroglia derived EVs could be a novel therapeutic strategy in countering chronic inflammation. Video Abstract.

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Autophagy-independent induction of LC3B through oxidative stress reveals its non-canonical role in anoikis of ovarian cancer cells.

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Autophagy-independent induction of LC3B through oxidative stress reveals its non-canonical role in anoikis of ovarian cancer cells.

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