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Activation of RAR? induces autophagy in SKBR3 breast cancer cells and depletion of key autophagy genes enhances ATRA toxicity.

ABSTRACT: All-trans retinoic acid (ATRA), a pan-retinoic acid receptor (RAR) agonist, is, along with other retinoids, a promising therapeutic agent for the treatment of a variety of solid tumors. On the one hand, preclinical studies have shown promising anticancer effects of ATRA in breast cancer; on the other hand, resistances occurred. Autophagy is a cellular recycling process that allows the degradation of bulk cellular contents. Tumor cells may take advantage of autophagy to cope with stress caused by anticancer drugs. We therefore wondered if autophagy is activated by ATRA in mammary tumor cells and if modulation of autophagy might be a potential novel treatment strategy. Indeed, ATRA induces autophagic flux in ATRA-sensitive but not in ATRA-resistant human breast cancer cells. Moreover, using different RAR agonists as well as RAR?-knockdown breast cancer cells, we demonstrate that autophagy is dependent on RAR? activation. Interestingly, inhibition of autophagy in breast cancer cells by either genetic or pharmacological approaches resulted in significantly increased apoptosis under ATRA treatment and attenuated epithelial differentiation. In summary, our findings demonstrate that ATRA-induced autophagy is mediated by RAR? in breast cancer cells. Furthermore, inhibition of autophagy results in enhanced apoptosis. This points to a potential novel treatment strategy for a selected group of breast cancer patients where ATRA and autophagy inhibitors are applied simultaneously.

SUBMITTER: Brigger D

PROVIDER: S-EPMC4558517 | biostudies-literature | 2015 Aug

REPOSITORIES: biostudies-literature

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Activation of RARα induces autophagy in SKBR3 breast cancer cells and depletion of key autophagy genes enhances ATRA toxicity.

Brigger D D Schläfli A M AM Garattini E E Tschan M P MP

Cell death & disease 20150827

All-trans retinoic acid (ATRA), a pan-retinoic acid receptor (RAR) agonist, is, along with other retinoids, a promising therapeutic agent for the treatment of a variety of solid tumors. On the one hand, preclinical studies have shown promising anticancer effects of ATRA in breast cancer; on the other hand, resistances occurred. Autophagy is a cellular recycling process that allows the degradation of bulk cellular contents. Tumor cells may take advantage of autophagy to cope with stress caused by ...[more]

PMID: 26313912

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Project description:Nucleoside reverse transcriptase inhibitors (NRTI), such as zidovudine (AZT), are constituents of HIV-1 therapy and are used for the prevention of mother-to-child transmission. Prolonged thymidine analogue exposure has been associated with mitochondrial toxicities to heart, liver, and skeletal muscle. We hypothesized that the thymidine analogue AZT might interfere with autophagy in myocytes, a lysosomal degradation pathway implicated in the regulation of mitochondrial recycling, cell survival, and the pathogenesis of myodegenerative diseases. The impact of AZT and lamivudine (3TC) on C2C12 myocyte autophagy was studied using various methods based on LC3-green fluorescent protein overexpression or LC3 staining in combination with Western blotting, flow cytometry, and confocal and electron microscopy. Lysosomal and mitochondrial functions were studied using appropriate staining for lysosomal mass, acidity, cathepsin activity, as well as mitochondrial mass and membrane potential in combination with flow cytometry and confocal microscopy. AZT, but not 3TC, exerted a significant dose- and time-dependent inhibitory effect on late stages of autophagosome maturation, which was reversible upon mTOR inhibition. Inhibition of late autophagy at therapeutic drug concentrations led to dysfunctional mitochondrial accumulation with membrane hyperpolarization and increased reactive oxygen species (ROS) generation and, ultimately, compromised cell viability. These AZT effects could be readily replicated by pharmacological and genetic inhibition of myocyte autophagy and, most importantly, could be rescued by pharmacological stimulation of autophagolysosomal biogenesis. Our data suggest that the thymidine analogue AZT inhibits autophagy in myocytes, which in turn leads to the accumulation of dysfunctional mitochondria with increased ROS generation and compromised cell viability. This novel mechanism could contribute to our understanding of the long-term side effects of antiviral agents.

Project description:AimFree fatty acid-induced lipotoxicity plays a crucial role in the progression of nonalcoholic fatty liver disease (NAFLD). In the present study we investigated the effects of a high-fat diet and free fatty acids on the autophagic process in hepatocytes in vivo and in vitro and the underlying mechanisms.MethodsLC3-II expression, a hallmark of autophagic flux, was detected in liver specimens from patients with non-alcoholic steatohepatitis (NASH) as well as in the livers of C57BL/6 mice fed a high-fat diet (HFD) up to 16 weeks. LC3-II expression was also analyzed in human SMMC-7721 and HepG2 hepatoma cells exposed to palmitic acid (PA), a saturated fatty acid. PA-induced apoptosis was detected by Annexin V staining and specific cleavage of PARP in the presence and absence of different agents.ResultsLC3-II expression was markedly increased in human NASH and in liver tissues of HFD-fed mice. Treatment of SMMC-7721 cells with PA increased LC3-II expression in time- and dose-dependent manners, whereas the unsaturated fatty acid oleic acid had no effect. Inhibition of autophagy with 3MA sensitized SMMC-7721 cells to PA-induced apoptosis, whereas activation of autophagy by rapamycin attenuated PA-induced PARP cleavage. The autophagy-associated proteins Beclin1 and Atg5 were essential for PA-induced autophagy in SMMC-7721 cells. Moreover, pretreatment with SP600125, an inhibitor of JNK, effectively abrogated PA-mediated autophagy and apoptosis. Specific knockdown of JNK2, but not JNK1, in SMMC-7721 cells significantly suppressed PA-induced autophagy and enhanced its pro-apoptotic activity; whereas specific knockdown of JNK1 had the converse effect. Similar results were obtained when HepG2 cells were tested.ConclusionJNK1 promotes PA-induced lipoapoptosis, whereas JNK2 activates pro-survival autophagy and inhibits PA lipotoxicity. Our results suggest that modulation of autophagy may have therapeutic benefits in the treatment of lipid-related metabolic diseases.

Project description:BackgroundPaclitaxel, a widely used chemotherapeutic drug, can induce apoptosis in variety of cancer cells. A previous study has shown preferential toxicity of paclitaxel to FLCN-deficient kidney cancer cell line, UOK257. In this report, we investigate the cellular and molecular mechanism of paclitaxel-induced autophagy and apoptosis in renal cancer cells with and without FLCN expression.MethodsTwo pairs of cell lines were used: FLCN siRNA-silenced ACHN cell line (ACHN-5968) and scrambled ACHN cell line (ACHN-sc); FLCN-null UOK257 cell line and UOK257-2 cell line restored with ectopic expression of FLCN. Autophagy was examined by western blot, GFP-LC3, transmission electron microscopy, and MDC assay. Cell viability and apoptosis were detected using MTT assay, DAPI stain and TUNEL assay. After inhibition of autophagy with 3-Methyladenine (3-MA) or Beclin 1 siRNA, cell viability and apoptosis were measured by MTT assay and TUNEL assay.ResultsAfter paclitaxel treatment, a dose-dependent decrease in cell viability and increase in apoptosis were observed in FLCN-deficient UOK257 and ACHN-5968 cells compared to their FLCN-expressing counterparts, suggesting that renal cancer cells without FLCN were more sensitive to paclitaxel. Enhanced autophagy was found to be associated with paclitaxel treatment in FLCN-deficient RCC cells. The MAPK pathway was also identified as a key pathway for the activation of autophagy in these kidney cancer cells. Inhibition of phosphorylated ERK with ERK inhibitor U0126 showed a significant decrease in autophagy. Furthermore, after inhibition of autophagy with 3-Methyladenine (3-MA) or Beclin 1 siRNA, apoptosis induced by paclitaxel was significantly increased in FLCN-deficient UOK257 and ACHN-5968 cells.ConclusionsPreferential toxicity of paclitaxel to FLCN-deficient kidney cancer cells is associated with enhanced autophagy. Suppression of autophagy further enhances paclitaxel-induced apoptosis in FLCN-deficient renal cancer cells. Our results suggest that paclitaxel combined with an autophagy inhibitor might be a potentially more effective chemotherapeutic approach for FLCN-deficient renal cancer.

Dataset Information

Activation of RAR? induces autophagy in SKBR3 breast cancer cells and depletion of key autophagy genes enhances ATRA toxicity.

Publications

Activation of RARα induces autophagy in SKBR3 breast cancer cells and depletion of key autophagy genes enhances ATRA toxicity.

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