Expression Of Autophagy Marker Proteins Research Articles

DDX53 Promotes Cancer Stem Cell-Like Properties and Autophagy.

Although cancer/testis antigen DDX53 confers anti-cancer drug-resistance, the effect of DDX53 on cancer stem cell-like properties and autophagy remains unknown. MDA-MB-231 (CD133+) cells showed higher expression of DDX53, SOX-2, NANOG and MDR1 than MDA-MB-231 (CD133−). DDX53 increased in vitro self-renewal activity of MCF-7 while decreasing expression of DDX53 by siRNA lowered in vitro self-renewal activity of MDA-MB-231. DDX53 showed an interaction with EGFR and binding to the promoter sequences of EGFR. DDX53 induced resistance to anti-cancer drugs in MCF-7 cells while decreased expression of DDX53 by siRNA increased the sensitivity of MDA-MB-231 to anti-cancer drugs. Negative regulators of DDX53, such as miR-200b and miR-217, increased the sensitivity of MDA-MB-231 to anti-cancer drugs. MDA-MB-231 showed higher expression of autophagy marker proteins such as ATG-5, pBeclin1Ser15 and LC-3I/II compared with MCF-7. DDX53 regulated the expression of marker proteins of autophagy in MCF-7 and MDA-MB-231 cells. miR-200b and miR-217 negatively regulated the expression of autophagy marker proteins. Chromatin immunoprecipitation assays showed the direct regulation of ATG-5. The decreased expression of ATG-5 by siRNA increased the sensitivity to anti-cancer drugs in MDA-MB-231 cells. In conclusion, DDX53 promotes stem cell-like properties, autophagy, and confers resistance to anti-cancer drugs in breast cancer cells.

Endurance exercise training induces fat depot-specific differences in basal autophagic activity

The purpose of this study was to uncover the effect of exercise training on the expression of autophagy marker proteins in epididymal white adipose tissue (eWAT), inguinal WAT (iWAT), and the stromal vascular fraction (SVF) collected from eWAT. Male Wistar rats aged 4–5 weeks were randomly divided into two groups, sedentary control (n = 7) and exercise-trained (n = 7). Rats in the exercise-trained group were exercised on a treadmill set at a 5° incline 5 days/week for 9 weeks. We determined that the expression levels of an autophagosome-associating form of microtubule-associated protein 1 light chain 3 (LC3)-II and of p62 were significantly higher in eWAT from exercise-trained than from control rats, while those of adipose-specific deletion of autophagy-related protein (ATG7) and lysosomal-associated membrane protein type 2A (LAMP2a) showed no difference between groups. However, in iWAT, the expression levels of LC3-II and ATG7 were significantly higher in exercise-trained than in control rats. The expression of p62 was highly correlated with that of peroxisome proliferator-activated receptor γ (PPARγ), a master regulator of adipogenesis and lipid metabolism, in both WAT types (eWAT, r = 0.856, P < 0.05; iWAT, r = 0.762, P < 0.05), whereas LC3-II and PPARγ levels were highly correlated in eWAT (r = 0.765, P < 0.05) but not in iWAT (r = −0.306, ns). In SVF, the expression levels of LC3II, ATG7, and LAMP2a were significantly higher in exercise-trained than in control rats. These results suggest that exercise training suppresses basal autophagy activity in eWAT, but that this activity is enhanced in iWAT and SVF collected from eWAT. Thus, the adaptation of basal autophagic activity following exercise training exhibits fat depot-specific differences.

P3-17-02: Targeting the Autophagy Pathway for Drug Resistance of Breast Tumor-Initiating Cells.

Abstract Background: Our clinical data and experimental evidence reveal that the tumor-initiating cells (TICs) in the original tumor are intrinsically resistant to conventional chemotherapy and radiation therapy and greatly enriched in residual breast cancers after such treatments. We have published a gene expression signature of such breast TICs, and our pathway analysis on the gene signature suggests that the activation of autophagy pathway may be an intrinsic characteristic of the TICs. This motivates us to further investigate the role of the autophagy pathway in TICs self-maintenance and their resistance to hormonal and chemo therapies, as well as their response to TIC-targeted therapies. Methods: A collective 84 well-documented autophagy pathway genes were used to compare the activation of autophagy pathway in different microarray datasets, 1) flow-sorted CD44+/CD24−/low cancer cells vs. all other cells (representing 20 patients), and cancer-derived MSs vs. corresponding primary bulk tumors (representing 17 patients), 2) before vs. after letrozole and doxetaxel treatments (representing 30 patient pairs), 3) before vs. after letrozole treatment (representing 176 patient pairs), and 4) before vs. after lapatinib treatment (representing 115 patient pairs). We applied the Significance Analysis of Microarrays (SAM) algorithm to analyze the expression data of 211 normalized probes for the 84 genes. Low-density RTPCR array for the 84 genes was used to confirm the differential expressed genes on tumor tissues of 18 letrozol treated patient pairs. Two human-cancer-in-mouse triple-negative xenograft tumor lines were treated with Notch pathway inhibitor alone or in combining with docetaxel. The tumor growth, mammosphere formation efficiency, and the expression of autophagy marker proteins were evaluated. Results: Overall, the enrichment analysis of the 84-gene set in all the above microarray datasets showed that the autophagy genes are significantly enriched in the differential expressed genes. In particular, 28 out of the 84 autophagy genes are significantly up-regulated in the TICs populations while 5 other autophagy genes are down-regulated with the false discovery rate (FDR) less than 0.05. For the letrozole and doxetaxel treatment microarray analysis, 34 out of the 84 autophagy genes are significantly up-regulated in the after-treatment group (p&amp;lt;0.01), and only 3 out of the 84 autophagy genes are down-regulated (FDR&amp;lt;0.05). Interestingly, significantly more autophagy genes (15 out of the 84) are down-regulated in the after-lapatinib treatment group (p&amp;lt;0.05), and less genes (13 out of 84) are up-regulated after lapatinib treatment, thus confirming previous observations that lapatinib may affect TICs. We confirmed these findings in two human cancer xenograft studies of Notch pathway inhibitor (GSI) to target TICs, which showed significantly reduced expressions of LC3 and p62, two autophagic markers. In both xenograft models, GSI reduced mammosphere formation efficiency compared to vehicle control (p&amp;lt;0.05). Conclusion: Activation of the autophagy pathway in TICs is a promising target to combat the drug resistance of breast cancer to conventional systemic therapy. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P3-17-02.

Abstract 4857: The role of autophagy in taxane resistant breast cancer models

Abstract Treatment with the taxanes (Paclitaxel or Docetaxel) is often the therapy of choice for women with advanced breast cancer. In most cases, the taxanes can arrest cell proliferation at the G2/M phase and cause cell death. However, some tumors develop resistance during the course of treatment, which is a major concern for both patients and physicians. Several mechanisms have been proposed to explain how resistance to the taxanes occurs, but this phenomenon remains incompletely understood. We hypothesize that changes in cell death signaling, through apoptotic and non-apoptotic pathways, are responsible for taxane resistance in breast cancer. Using the MDA-MB-231 breast cancer cell line model, we selected cells that are resistant to either Docetaxel (DocR) or Paclitaxel (PacR). Subsequently we studied levels of cell death and the response to IC50 concentrations of these drugs. Given the molecular crosstalk that can occur between cells dying by apoptosis and the autophagy process, Western blots for prosurvival Bcl-2 and NFκB were performed, and these showed reduced expression in the taxane resistant cells compared to the sensitive cell lines. In conjunction with changes in apoptotic markers in the resistant cell lines, there were also changes in autophagy protein markers such as increased expression of cleaved LC3 and a decrease in p62/SQSTM1 expression, particularly in the PacR cells. Beclin-1 and XBP-1 expression, however, were not differentially expressed between the sensitive and both resistant cell lines. Beclin-1 and p62 have been implicated in the regulation of both Bcl-2 and NFκB expression. In order to determine whether the changes in autophagy marker protein expression was an indication of an alternative cell death process or cell survival, proliferation assays were performed with sensitive and resistant cells. Cells were counted after exposure for 7 days with and without 3-Methyladenine, an inhibitor of autophagosome formation. The addition of 3-MA significantly decreased cell number suggesting that autophagy may be a mechanism of cell survival in both taxane resistant models. Given our preliminary data, we propose to further investigate the role of autophagy and other alternative cell death evasion pathways seen in MDA-MB-231 taxane resistant and sensitive breast cancer cells. Our studies aim to improve the understanding of how different mechanisms of cell death evasion contribute to the resistance phenotype. Ultimately our studies may not only improve our understanding of taxane resistance at the bench, but may also provide the framework for formulation of novel therapies that may improve the success of taxane therapy in women with breast cancer in the clinic. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4857.