TRP14Induces Paclitaxel Resistance In Ovarian Cancer Via Modulating Cell Autophagy In Vitro And In Vivo

Ovarian cancer is the most lethal disease among all gynecological malignancies and the5-year survival from the disease is only44%for all stages and27%for advanced stages. First-line chemotherapy with paclitaxel and platinum combination has yielded responses rates of more than80%since the1990’s. However, most patients with ovarian cancer are relapsed in2-3years after initial treatment and almost all of the recurred patients are resistant to chemotherapy. Chemo-resistance remains one of the essential reasons for treatment failure and death associated with ovarian cancer.Paclitaxel is a microtubule-stabilizing drug that causes mitotic arrest and leads to cell death. Various evidences have shown that paclitaxel resistance is a process with multifactorial participation that may originate through a series of modification, including the overexpression of multidrug transporter P-glycoprotein that pumps paclitaxel out of tumor cells, the influence of the composition of βtubulin isotypes or the changes of microtubule dynamics, modulation of paclitaxel-induced apoptosis by pro-or anti-survival regulators such as Bcl2family proteins, and others. However, the progresses on reversing paclitaxel resistance are limited according to the possible mechanisms.Recent studies have revealed an association between autophagy and drug resistance. Chemotherapy agents, including paclitaxel, may lead to an autophagic response. Autophagy, one of the programmed cell death manners, is discovered in1960’s and also an evolutionarily conserved process involved in maintaining cellular homeostasis and metabolic balance. Recent studies have shed light on the functional role of autophagy in cancers. According to the mode of cargo delivery to the lysosome, three forms of autophagy have been identified:chaperone-mediated autophagy, microautophagy, and macroautophagy. Macroautophagy (herein referred to as autophagy) is characterized by sequestration of bulk cytoplasm, long-lived proteins and cellular organelles in double-membrane vesicles, called autophagosomes, which are ultimately delivered to and degraded in the lysosomes and recycling. This process is precisely regulated and sequentially involves Atgl/Atg13/Atg17complex, Vps34/Beclinl/Atgl4complex, Atg5/Atgl2/Atgl6complex, LC3, and others. Of which, Beclinl plays a key role in autophagy regulation. In light of different circumstance and stressful conditions, autophagy may induce programmed cell death or promote cell survival. Under stress such as nutrition deprivation or drug treatment, autophagy could provide nutrition or get rid of damaged proteins or organelles through autophagic catabolism, and maintain cell survival. However, sustained autophagy may eventually lead to cell death when prolonged stress overwhelms the turnover capacity of cells. Recent studies have implied that autophagy is not only the manner of cell death after chemotherapy, but also one of the mechanisms of chemo-resistance. It will exploit a new field of overcoming ovarian cancer if the regulation of autophagy in paclitaxel resistance has been illustrated.To further elucidate a novel potential mechanism of paclitaxel resistance in ovarian cancer, we applied Liquid Chromatography-Mass Spectrometry/Mass Spectrometry-based label free quantitative proteomic approach to reveal differentially expressed proteins between paclitaxel resistant SKOV3-TR30and parental SKOV3cells. We found a substantially raised expression of Thioredoxin-related protein14(TRP14) in SKOV3-TR30cells through qRT-PCR and Western Blot validation. TRP14is a novel14-kDa disulfide reductase and consists of123amino acids. This protein shares20%sequence identity and37%similarity with human Trx1(105amino acid residues). So TRP14is a new member of Thioredoxin family with two active site cysteine residues (Cys43and Cys46) in its CPDC motif. The biological function of TRP14is rarely understood, but it was recently found that TRP14was involved in TNF-asignaling pathway. A number of studies have demonstrated a role of TNF-a in stimulating autophagy in human cells, including macrophages, vascular smooth cells, skeletal muscle cells, T lymphoblastic leukaemic cells, Ewing sarcoma cells and breast cancer cells. Thus, there may be a link among TRP14overexpression, autophagy and paclitaxel resistance in ovarian cancer.In this study, we firstly found that paclitaxel induced upregulation of TRP14, formation of autophagosome and increased autophagy flux using Western Blot detection, GPF-LC3plasmid transfection and Transmission electron microscopy. Beclinl abrogation with siRNA suppressed cell autophagy and potentiated paclitaxel sensitivity in ovarian cancer cells. Moreover, TRP14manipulation with siRNA interference and plasmid expression system altered Beclinl expression, cell autophagy, and paclitaxel sensitivity correspondingly in ovarian cancer cells. TRP14and Beclinl expression were detected in157ovarian cancer tissues by immunohistochemical staining. High expression of RP14and Beclinl were significantly associated with chemo-resistance, high risk clinicopathological parameters and poorer prognosis. So TRP14promotes paclitaxel resistance via Beclinl participation in ovarian cancer and may be use as predictor of chemotherapy response and prognosis in ovarian cancer. Part I Paclitaxel induces autophagy and leads to paclitaxel resistance in ovarian cancer cellsObjective:To evaluate the influence of paclitaxel on autophagosome formation and autophagy flux in ovarian cancer cells. To confirm the effect of autophagic response on paclitaxel sensitivity in ovarian cancer cells.Methods:To evaluate the influence of paclitaxel on autophagosome formation, amounts of LC3B、Atg5and Beclinl were detected by Western Blot, GFP-LC3-II positive puncta were counted through fluorescence microscopy and the ultra-structure of autophagosomes were identified through transmission electron microscopy after ovarian cancer cells were exposed to various concentration of paclitaxel. To value the autophagy flux, LC3B-II turnover and p62accumulation were detected by Western Blot after ovarian cancer cells treated with paclitaxel plus BafAl or not. Paclitaxel sensitivity was assessed after Beclinl expression was abrogated using siRNA in ovarian cancer cells.Results:1. The amounts of LC3B-IL Atg5and Beclinl were substantially increased after exposure to paclitaxel at various concentrations in A2780,3AO and SKOV3cells.2. The numbers of GFP-LC3-II positive puncta per cell were significantly increased after24-hour exposure to paclitaxel in A2780,3AO and SKOV3cells which were identified through transmission electron microscopy.3. The accumulation of LC3B-Ⅱ and p62were larger after paclitaxel treatment compared with no paclitaxel treatment in ovarian cancer cells.4. Autophagy and Paclitaxel resistance were decreased after Beclinl expression was abrogated using siRNA interference.Conclusions:1. Paclitaxel pomotes formation of autophagosome and increases autophagy flux in ovarian cancer cells.2. Autophagic response induced by paclitaxel leads to drug resistance in ovarian cancer cells. Part Ⅱ TRP14modulates autophagy and paclitaxel sensitivity via Beclinl participationObjective:To illustrate that TRP14modulates autophagy and paclitaxel sensitivity via Beclinl participationMethods:Alteration of TRP14expression was detected by Western Blotwhen when ovarian cancer cells were treated with paclitaxel. The expression of Beclinl, LC3-II, Atg5, as well as autophagy and paclitaxel sensitivity were assessed after TRP14manipulation using siRNA or pcDNA3.1(+)-TRP14plasmid.Results:1. Paclitaxel induced upregulation of TRP14in A2780,3AO and SKOV3cells2. When TRP14was knocked down by siRNA, the levels of LC3-II, Atg5and Beclinl proteins were decreased in A2780and3AO cells, On the contrary, LC3-II, Atg5and Beclinl levels were increased in A2780and3AO cells that were enforcedly expressed with pcDNA3.1(+)-TRP14plasmid. Moreover, the induction of autophagy by TRP14overexpression was suppressed by Beclinl compromise.3. TRP14knockdown significantly increased the sensitivity to paclitaxel in ovarian cancer cells. Reversely, TRP14overexpression conferred protection from paclitaxel for A2780and3AO cells. The cytoprotection owning to TRP14overexpression was reversed by Beclinl knockdown. Conclusions:1. TRP14plays important role on paclitaxel resistance in ovarian cancer.2. TRP14modulates autophagy and paclitaxel sensitivity via Beclinl participation. Part Ⅲ The high expression of TRP14and Beclinl proteins in ovarian cancer tissues is associated with poorer patient prognosisObjective:To establish the relationship between the expression of TRP14and Beclinl with clinical prognosis in ovarian cancer.Methods:We detected TRP14expression in157ovarian cancer tissues by immunohistochemical staining. The relationships between the staining of TRP14and Beclinl and clinicopathological parameters, such as age, FIGO stage, tumour grade, ascitic fluid volume, serum CA125, primary surgery, chemo-resistance, prognosis, were analyzed.Results:1. TRP14high expression was significantly correlated with chemo-resistance, higher level of serum CA125, suboptimal primary surgery and poorer PFS and OS.2. Beclinl high expression was significantly correlated with chemo-resistance, advanced FIGO stage, higher level of serum CA125, suboptimal primary surgery, poorer PFS and OS.3. The high expression of TRP14was significantly associated with high expression of Beclinl.Conclusions:1. High expression of RP14and Beclinl were significantly associated with chemo-resistance, high risk clinicopathological parameters and poorer prognosis.2. TRP14and Beclinl may be use as predictor of chemotherapy response and prognosis in ovarian cancer...