Proteamic-based Discovery Of Redox-related Genes Mediate Drug Resistance In Prostate Cancer Cells

Prostate cancer is the most common malignancy in male. In recent years confirmed PCa, especially for advanced or metastatic PCa, represents an obviously rising trend in China with the aging and the diet habit changing. Endocrine therapy is the preferred treatment for PCa, but most of the patients will relapse one year after treatment, evolved into hormone refractory prostate cancer (HRPC), often accompanied by bone metastasis, high degree of malignancy. At present, docetaxel based chemotherapy is the first-line regimen in the treatment of HRPC, can elevate the survival rate of patients. However, chemotherapy drug side effects and drug resistance are seriously restricts its therapeutic effect. Several molecular mechanisms have been proposed to drive the development of docetaxel resistance in CRPC including increases in the expression levels of α-and β-tubulin, ATP-binding cassette (ABC) drug transporters such as P-glycoprotein and multidrug resistance-associated protein-1(MRP1), and anti-apoptotic proteins of Bcl-2, XIAP, Survivin, and Clusterin. In addition, inflammatory mediators interleukin (IL)-1β, IL6, and tumor necrosis factor (TNF)-a that are present in the microenvironment of many types of cancer are believed to contribute to PCa metastasis and drug resistance. However, prostate cancer multi-drug resistant cells are special, such as no expression of P-gp, while IL6expression was high. However, targeting individual genes, such as suppression of Bcl-2with antisense oligonucleotide, or targeting IL6by antibody yields limited clinical impact in PCa. Therefore, understanding of the insights of drug resistant mechanisms and identification of central factors that control multiple downstream genes related to phenotype alterations in drug resistance would be an attractive approach to minimize the docetaxel resistance in PCa.Our research group found that, in the multi-drug resistant cells (PC3/Doc) induced by docetaxel from prostate cancer PC3cells, microtubule protein increased, inflammation factor IL6was upregualted significantly, but no expression of P-gp. To determine the mechanism of drug resistance, this study used proteomics, analysised the different protein expression between PC3and PC3/Doc cells and identified ten proteins. SOD2was upregulated significantly in PC3/Doc cells. Futhermore, our recent data suggest PC3/Doc cells demonstrate a redox reducing compared to PC3cells. The proteins regulated by redox state such as insulin-like growth factor1receptor (IGF-1R),(B-cell CLL/lymphoma2) BCL2increased. Further study revealed that SOD2acted as a negative regulator of β-arrestinl that is an important adaptor responsible for degradation of IGF-1R via the changes in ROS. In addition, inflammatory factor IL6increased IGF-1R mRNA levels by up regulating the expression of STAT3, IL6also can up regulate the expression of SOD2.Therefore, the redox state of the cell (low levels of ROS) and inflammatory cytokines (high expression of IL6) upregulated IGF-1R in the protein expression and transcriptional level, respectively. The high expression of IGF-1R participated in drug resistance by up regulating the expression of BCL2and multidrug resistance associated protein drug transporter2(MRP2). Part I Identification of SOD2by proteomics acts as a chemoresistance modulator一、The establishment of PC3docetaxel resistance cell lineProstate cancer androgen independent cell lines PC3cells have a strong anti apoptosis ability and high degree of malignancy. PC3/Doc cells were selected by growing the PC3cells in increasing concentrations of docetaxel, until the cells were freely dividing in20nM docetaxel medium.The IC50for docetaxel in the PC3/Doc cells was38-fold higher than that in PC3cells, for doxorubicin were8-fold higher. But cisplatin resistance in the PC3/Doc cells is weak (resistance index was2times) when compared to the parental cells. PC3/Doc cell acquired multidrug resisttance.二、Proteomics-based analysis of differentially expressed proteins in PC3and PC3/Doc cells1. Protein expression difference spectrum between PC3cells and multidrug resistant cell PC3/Doc was analysised by Fluorescent difference gel electrophoresis and mass spectrometry:Through analysis of the48differences, we identified10differences in protein expression.5proteins were up-regulated:superoxide dismutase2(SOD2), heat shock protein90β1(HSP90B1), proliferating cell nuclear antigen (PCNA), two protein disulfide isomerase A3(PDIA3), cytochrome b-cl complex subunit1(QCR1);5protiens were down-regulated:perilipin3(PLIN3), Ras specific GTPase binding protein (RANG), type II keratin (K2C7), Nucleoside diphosphate kinase A (NDKA), vimentin (VIME).2. Verification of the differential expression protein:Differentially expressed proteins screened by proteomics were tested by western blot. The results showed that SOD2, HSP90B1, PCNA, PDIA3were upregulated in resistant cells, while NDKA and VIME were down-regulated in resistant cells, consistent with the proteomics. In addition, our previous Microarray data show that, the expression of SOD2, HSP90, PDIA3is also higher at the transcriptional level in the resistant cells.3. Function analysis of the differential expression protein:According to the experimental conditions, RNAi method was used to reduce expression of SOD2, HSP90B1and PDIA3in multidrug resistance cells, and sensitivity of cells to docetaxel were measured. The results show that knock down of SOD2expression increased the sensitivity of cells to docetaxel significantly. Over-expression of SOD2in PC3cells enhanced the tolerance to docetaxel. In consideration of the expression and function of SOD2, we confirmed that SOD2play an important role in docetaxel resistance in prostate cancer cells and further analysis of mechanism of its participation in drug resistance.Part II SOD2-mediated chemoresistance is associated with enhanced IGF-1R in docetaxel resistant cells一、The effect of SOD2etc on cell redox state:1. Reduced ROS in docetaxel resistant cells:Flow cytometry analysis showed that, compared with the susceptible strain PC3cells, the level of ROS in PC3/Doc cells decreased, ratio of reduced GSH and oxidized glutathione GSSG rised.2. SOD2participate in the regulation of ROS level:Flow cytometry analysis showed, decrease the expression of SOD2in resistant cells will increased level of ROS, demonstated that high expression of SOD2can partially reduce ROS. The results of quantitative PCR and Microarray data show that, other antioxidant enzymes or proteins, such as catalase (CAT), glutathione peroxidase4(GPX4), glutathione S-transferase (GSTP1), thioredoxin (TXN) was significantly upregualted in the resistant cells, while Superoxide dismutase (SOD1), thioredoxin peroxidase3(PRDX3), nuclear factor erythroid2-like2(NFE2L2) increased slightly. The above results showed that the combined action of a series of anti oxidase resulted in reduced ROS, and SOD2involved in. 二、SOD2positively regulates IGF-1R protein, but has no effect on IGF-1R transcription1. Redox regulation of gene expression:Using the Microarray data analysis of the expression of26genes regulated by redox state, combined with quantitative PCR verification, we found that the expression of IGF-1R, CXCR4, BCL2were upregulated in resistant cells. Western blot experiments further established that IGF-1R and BCL2were highly expressed in resistant cells2. SOD2upregulated IGF-1R protein expression:Depletion of SOD2in resistant PC3/Doc cells, IGF-1R protein abundance was significantly impaired, but not its mRNA. Conversely, forced expression of SOD2in sensitive PC3cells predominantly caused an increased protein level of IGF-1R, while mRNA level of IGF-1R remained unaffected compared with the cells transfected with control vector.三、SOD2impairs P-arrestinl-mediated degradation of the IGF-1R protein in resistant cells1. The stability of IGF-1R increased in the resistant cells:Consideration of upregulation of IGF-1R protein, we first analyze the protein stability. Ubiquitination and subsequent degradation of IGF-1R are usually mediated by ARRB1and MDM2or NEDD4and GRB10. Quantitative PCR results showed that the expression of NEDD4and GRB10are the same in the sensitive cells and resistant cells, but the expression of ARRB1in drug-resistant cells decreased significantly, MDM2increased significantly. Western blot results showed that, the protein level of ARRB1in resistant cells also decreased significantly. As an adaptor protein, ARRB1recruit MDM2(E3) to IGF-1R, which is crucial for the degradation of IGF-1R. Knock down of ARRB1in PC3cells restored the IGF-1R expression, which show that ARRB1mediated degradation of IGF-1R in PCa cells.2. Knockdown of SOD2in resistant cells resulted in up-regulation of β-arrestinl, associated with declined IGF-1R. Forced expression of SOD2in sensitive cells led to decreased β-arrestinl, and significant up-regulation of the receptor.3. Addition of an anti-oxidant agent vitamin C (VC) time-dependently abolished β-arrestinl production and polyubiquitinated IGF-1R in sensitive cells, while expressions of β-arrestinl and polyubiquitinated IGF-1R were noticeably rescued under conditions where ROS was induced by H2O2in resistant cells.4. Transcriptional regulation of.ARRB1:Combination of software prediction and gene chip, we predict that ARRB1promoter contained transcription factors FOXP1, FOXA1and YY1binding sites, and the expression of these transcription factors decreased based microarray data. Luciferase-based assays show that, transfection of the expression plasmid FOXP1can enhance the activity of the ARRBl promoter both in PC3and PC3/Doc cells, which proved that FOXPl can promote the transcription activity of ARRB1.5. The effect of ROS on IGF-1R protein stability was confirmed on animal model: The PC3cells were injected into nude mice and formed tumor, and tail vein injection of VC solution. Compared with placebo-treated control, VC treatment promoted tumor growth as indicated by increased tumor weight. Tumor cell proliferation was also confirmed with the increased Ki67positive cells in VC-treated animals. Consistent with the observations in culture cells, VC treatment significantly impaired β-arrestinl expression, accompanied with upregulation of the IGF-1R.四、IL6stimulates IGF-1R transcription and reduces the receptor degradation via regulations of SOD2and P-arrestinl in resistant cells1. IL6was up-regulated in PC3/Doc cells:Microarray data suggests the expression of IL6resistant cells increased significantly, ELISA result further confirmed that compared with the PC3cells, IL6expressed in drug-resistant cell PC3/doc increases significantly.2. IL6regulated IGF-1R through SOD2:Following blockade of IL6in resistant cells, SOD2level was significantly abrogated, whereas accumulation of β-arrestinl was markedly evident and simultaneously caused elimination of IGF-1R.2. IL6regulated IGF-1R on transcripation level:qPCR results indicated that blocking IL6activity suppressed mRNA level of IGF-1R in resistant cells, suggesting a role of IL6in transcriptional regulation of IGF-1R. Transcription factor STAT3, an important downstream target of IL6, was activated in resistant cells compared to sensitive cells. Rendered IL6by targeting antibody decreased phosphor-STAT3and IGF-1R and depletion of STAT3affected IGF-1R mRNA expression level as well. Luciferase-based assays showed that activation of IGF-1R promoter that contains STAT3binding sequence by co-transfection of STAT3expression plasmid was observed in cells.Part III The mechanism of IGF-1R participating in docetaxel resistance in prostate cancer一、High expression of IGF-1R participated in drug resistance though activating its mediated signaling pathways1. IGF-1R is activated in PC3/Doc cells:Microarray chip date show that compared with PC3cells, IGF-1R ligands such as IGF-Ⅰ, IGF-Ⅱ have no change in PC3/Doc cells. However, IGF-1R was upregulated and sustained activated in PC3/Doc cells. The phosphor-IGF1R (Tyr980/1131/1135/1136) increased and downstream target gens, such as the phoshpor-Akt and phosphor-Shc were activated.2. IGF-1R participated in drug resistance:Knock down of IGF-1R in PC3/Doc cells enhanced the sensitivity to docetaxel.二、IGF-1R participated in drug resistance thongh regulating MRP2and BCL21. MRP2and BCL2were upregulated in PC3/Doc cells:There were reports that MRP2and BCL2were regulated by IGFIR. Western blot show that the expression of MRP2and BCL2increased in PC3/Doc cells.2. IGF-1R regulates MRP2and BCL2through downstream signaling pathways: IGF-1R works in two ways:one is through the downstream signaling pathways; IGF-1R can translocate to the nucleus from the cell membrane, act as an transcription factor or cofactor. Immunofluorescence and Western blotting results show that, there is no IGF-1R expression in the nucleus of PC3/Doc cells. We mutated three lysine (1025,1100,1120) to arginine of IGF-1R, which resulted in IGF-1R cannot be sumolization and cannot translocate to nucleus. The plasmids pcDNA3.1-IGF-1R-WT (wild type) and pcDNA3.1-IGF-1R-M were transfected into PC3cells, resulted in upregulation of MRP2and BCL2. However, there was no significant difference between wild type IGF-1R and mutant IGF-1R on the exprreson of MRP2and BCL2. This demonstrated that IGF-1R regulated MRP2and BCL2through downstream signaling pathways, not by translocate to nucleus.3. Other resistance related protein function:P-gp or MRP2and CYP protein were found to be the key molecular in docetaxel pharmacokinetics. MRP was regulated by IGF-1R. We wonder whether CYP can be regulated by IGF-1R. Microarray and quantitative PCR results showed that CYP3A4, CYP3A5, CYP3A7and CYP3A43in resistant cells were upregulated with varying degrees. However, knock down of IGF-1R has no significant effect on the expression of CYPs. This demonstrated CYPs may participate in drug resistance in Prostate caner cells, but no were regulated by IGF-1R.Part IV Conclusions and Innovation一、Conclusions1. DIGE/MOF/MS proteomics analysis showed that high expression of SOD2in human prostate cancer multidrug resistant cells particiapate in docetaxel resistant.2. SOD2acted as a negative regulator of β-arrestinl that was an important adaptor responsible for degradation of IGF-1R via the changes in ROS, positively regulated IGF-1R, but did not affect the mRNA of IGF-1R.3. IL6positively regulates IGF-1R:at transcriptional level, IL6promote IGF-1R transcription by STAT3; at protein post-translational level, IL6positive regulates IGF-1R.through the regulation of SOD2and ARRB1.4. High expression of IGF-1R in drug-resistant cells was sustained activated, participated in multidrug resistance, through up regulation of BCL2and MRP2.二、Innovation and defects1. We firstly reported that SOD2was upregulated significantly in prostate cancer multidrug resistance cells, linking SOD2to docetaxel resistant. We firstly revealed that SOD2positively regulates IGF-1R though ROS and ARRB1. Whether there are other mechanisms which explain the relationship between SOD2and docetaxel resistance are not clear.2. We firstly reported IL6positively regulated IGF-1R through the transcription factor STAT3. IL6can up regulate the expression of SOD2, the latter increased the protein level of IGF-1R by ROS. Whether there were any other transcriptional factors regulating the expression of IGF-1R and How IL6regulate the expression of SOD2need futher study.3. We firstly reported that the low expression of ARRB1in prostate cancer multidrug resistance cells negatively regulated drug resistance. We firstly reported that ROS positively regulated the expression of ARRB1, and IL6negatively regulated the expression of ARRB1. But the mechanism of ROS, IL6regulating ARRB1expression, and mechanism of lower level of ARRB1participated in the process of drug resistance are required further reseach.