Development Of Docetaxel Resistance Research Articles

Polysaccharides from Lentinus edodes prevent acquired drug resistance to docetaxel in prostate cancer cells by decreasing the TGF-β1 secretion of cancer-associated fibroblasts.

Prostate cancer is one of the most prevalent lethal diseases among men globally. In the treatment of prostate cancer, the limited therapeutic efficacy of the standard non-hormonal systemic therapy docetaxel (DTX) represents an important challenge. Cancer-associated fibroblasts (CAFs) play a crucial role in resistance to therapy because of their prevalence and functional pleiotropy in tumor environments. Our previous research revealed that MPSSS, a novel polysaccharide extracted from Lentinus edodes, could significantly attenuate the immunosuppressive function of myeloid suppressor cells and CAFs. In this study, we investigated whether MPSSS could potentiate the efficacy of DTX against prostate cancer by inhibiting CAF-induced chemoresistance and elucidated its underlying mechanisms. The sensitivity of PC-3 prostate cancer cells cultured with conditioned medium derived from CAFs (CAF-CM) to DTX was assessed. The resistance effect induced by CAF-CM was abolished when CAFs were pretreated with MPSSS. Bioinformatic analysis of datasets from the Gene Expression Omnibus database revealed the activation of the transforming growth factor β1 (TGF-β1) signaling pathway in DTX-resistant cells. Based on this finding, we demonstrated that treatment with the TGF-β1 receptor inhibitor SB525334 reversed DTX resistance in CAFs, suggesting that TGF-β1 secreted by CAFs was a crucial intermediary in the development of DTX resistance in PC3 cells. Further research revealed that MPSSS decreases the secretion of TGF-β1 by inhibiting the JAK2/STAT3 pathway via Toll-like receptor 4 in CAFs. Overall, MPSSS might be a potential adjuvant treatment for DTX resistance in prostate cancer.

Docetaxel Resistance in Castration-Resistant Prostate Cancer: Transcriptomic Determinants and the Effect of Inhibiting Wnt/β-Catenin Signaling by XAV939.

Castration-resistant prostate cancer (CRPC) is a common form of prostate cancer in which docetaxel-based chemotherapy is used as the first line. The present study is devoted to the analysis of transcriptome profiles of tumor cells in the development of resistance to docetaxel as well as to the assessment of the combined effect with the XAV939 tankyrase inhibitor on maintaining the sensitivity of tumor cells to chemotherapy. RNA-Seq was performed for experimental PC3 cell lines as well as for plasma exosome samples from patients with CRPC. We have identified key biological processes and identified a signature based on the expression of 17 mRNA isoforms associated with the development of docetaxel resistance in PC3 cells. Transcripts were found in exosome samples, the increased expression of which was associated with the onset of progression of CRPC during therapy. The suppression of pathways associated with the participation of cellular microtubules has also been shown when cells are treated with docetaxel in the presence of XAV939. These results highlight the importance of further research into XAV939 as a therapeutic agent in the treatment of CRPC; moreover, we have proposed a number of mRNA isoforms with high predictive potential, which can be considered as promising markers of response to docetaxel.

Molecular Profiling of Docetaxel-Resistant Prostate Cancer Cells Identifies Multiple Mechanisms of Therapeutic Resistance.

Simple SummaryTherapeutic options for the treatment of men with metastatic castration-resistant prostate cancer are limited. Docetaxel—a taxane-based chemotherapeutic agent—was the first treatment to demonstrate significant efficacy in the treatment of this disease. However, responses to docetaxel are frequently curtailed by development of drug resistance, and patients eventually succumb to disease progression due to acquisition of drug resistance. In this study, we established drug-resistant prostate cancer cell lines and identified several mechanisms that may be associated with the development of drug resistance in prostate cancer. Actioning these mechanisms could provide a potential approach to re-sensitize drug-resistant cancer cells to docetaxel treatment and thereby further add to the life-prolonging effects of this drug in men with metastatic castration-resistant prostate cancer.Docetaxel—a taxane-based chemotherapeutic agent—was the first treatment to demonstrate significant improvements in overall survival in men with metastatic castration-resistant prostate cancer (mCRPC). However, the response to docetaxel is generally short-lived, and relapse eventually occurs due to the development of resistance. To explore the mechanisms of acquired docetaxel resistance in prostate cancer (PCa) and set these in the context of androgen deprivation therapy, we established docetaxel-resistant PCa cell lines, derived from the androgen-dependent LNCaP cell line, and from the LNCaP lineage-derived androgen-independent C4-2B sub-line. We generated two docetaxel-resistant LNCaPR and C4-2BR sub-lines, with IC50 values 77- and 50-fold higher than those of the LNCaP and C4-2B parental cells, respectively. We performed gene expression analysis of the matched sub-lines and found several alterations that may confer docetaxel resistance. In addition to increased expression of ABCB1, an ATP-binding cassette (ABC) transporter, and a well-known gene associated with development of docetaxel resistance, we identified genes associated with androgen signaling, cell survival, and overexpression of ncRNAs. In conclusion, we identified multiple mechanisms that may be associated with the development of taxane drug resistance in PCa. Actioning these mechanisms could provide a potential approach to re-sensitization of docetaxel-resistant PCa cells to docetaxel treatment and thereby further add to the life-prolonging effects of this drug in men with mCRPC.

Endothelial Cells Promote Docetaxel Resistance of Prostate Cancer Cells by Inducing ERG Expression and Activating Akt/mTOR Signaling Pathway.

Docetaxel is a first-line chemotherapy for the treatment of patients with castration-resistant prostate cancer (CRPC). Despite the good initial response of docetaxel, drug resistance will inevitably occur. Mechanisms underlying docetaxel resistance are not well elaborated. Endothelial cells (ECs) have been implicated in the progression and metastasis of prostate cancer. However, little attention has been paid to the role of endothelial cells in the development of docetaxel resistance in prostate cancer. Here, we sought to investigate the function and mechanism of endothelial cells involving in the docetaxel resistance of prostate cancer. We found that endothelial cells significantly promoted the proliferation of prostate cancer cells and decreased their sensitivity to docetaxel. Mechanistically, basic fibroblast growth factor (FGF2) secreted by endothelial cells leads to the upregulation of ETS related gene (ERG) expression and activation of the Akt/mTOR signaling pathway in prostate cancer cells to promote docetaxel resistance. In summary, these findings demonstrate a microenvironment-dependent mechanism mediating chemoresistance of prostate cancer and suggest that targeting FGF/FGFR signaling might represent a promising therapeutic strategy to overcome docetaxel resistance.

DNA polymerase theta repression enhances the docetaxel responsiveness in metastatic castration-resistant prostate cancer

ObjectiveDocetaxel remains a main treatment for metastatic castration-resistant prostate cancer (mCRPC); however, the development of docetaxel resistance has been found in some mCRPC patients. The aim of this work is to identify an effective biomarker for predicting therapeutic effectiveness of docetaxel in mCRPC patients. MethodsWe examined DNA polymerase theta (POLQ) expression in The Cancer Genome Atlas (TCGA) database and Tissue microarray. Kaplan-Meier analyses were performed to estimate the prognostic significance of POLQ. A series of functional analyses were conducted in cell lines and xenograft models. Regulated pathways were predicted by Geneset Enrichment Analysis (GSEA) software and further investigated by luciferase reporter and RT-PCR assays. ResultsWe found that POLQ mRNA levels in CRPC tissues was significantly higher than that of other DNA polymerases in non-CRPC prostate tissues. POLQ upregulation was extensively detected in mCRPC and strongly predicted a poor prognosis. POLQ knockdown enhanced docetaxel sensitivity in a cell-based cytotoxicity assay and promoted the therapeutic effect on the tumor growth of metastatic PC-3M cells in xenograft models. The computational simulation by GSEA software significantly predicted the association between POLQ upregulation and the activation of E2F/G2M checkpoint-related pathways. Moreover, luciferase reporter and RT-PCR assays demonstrated that POLQ knockdown downregulated the transcriptional regulatory activity of E2F and repressed E2F/G2M checkpoint-regulated CDK1 in mCRPC cells. ConclusionOur results suggest that POLQ serves as a predictive factor for poor docetaxel response and provide a novel strategy to enhance the anticancer effects of docetaxel therapy by targeting POLQ in mCRPC patients.

Identification of biomarker microRNA-mRNA regulatory pairs for predicting the docetaxel resistance in prostate cancer.

Background: Docetaxel resistance is a cursing problem with adverse effects on the therapeutic efficacy of prostate cancer (PCa), involving interactions among multiple molecular components. Single or limited molecules are not strong enough as prediction biomarkers of drug resistance. Network biomarkers are considered to outperform individual markers in disease characterization.Methods: In this study, key microRNAs (miRNAs) as biomarkers were identified from the PubMed citations and miRNA expression profiles. Targets of miRNAs were predicted and enriched by biological function analysis. Key target mRNAs of the biomarker miRNAs were screened from protein-protein interaction network and gene expression profiles, respectively. The results were validated by the assessment of their predictive power and system biological analysis.Results: With this approach, we identified 13 miRNAs and 31 target mRNAs with 66 interactions in the constructed network. Integrative functional enrichment analysis and literature exploration further confirmed that the network biomarkers were highly associated with the development of docetaxel resistance.Conclusions: The findings from our results demonstrated that the identified network biomarkers provide a useful tool for predicting the docetaxel resistance and may be helpful for serving as prediction biomarkers and therapeutic targets. However, it is necessary to conduct biological experiments for further investigating their roles in the development of docetaxel resistance.

Targeting the androgen receptor (AR) with AR degradation enhancer ASC-J9® led to increase docetaxel sensitivity via suppressing the p21 expression

Chemotherapy with docetaxel remains the effective therapy to suppress castration resistant prostate cancer (CRPC) in some patients. However, most chemotherapy with docetaxel eventually fails with the development of docetaxel resistance after 18-weeks of treatment. Here we found docetaxel treatment might have an adverse effect of increasing the androgen receptor (AR) protein level in the CRPC cells, and combining docetaxel with anti-AR therapy using AR-shRNA or the AR degradation enhancer ASC-J9® may increase docetaxel sensitivity to better suppress the CRPC cell growth. Mechanism dissection found docetaxel might have the adverse effect of increasing the AR protein stability via suppressing the AR ubiquitination due to the increased AR phosphorylation. The consequence of such increased AR protein may then lead to increase p21 expression via transcriptional regulation. Preclinical studies with in vitro cells lines also demonstrated that targeting AR with ASC-J9® led to suppressing the AR-increased p21 expression to improve the docetaxel sensitivity in the CRPC cells that already developed docetaxel resistance. Together, these results suggest that a combined therapy of docetaxel and ASC-J9® is a novel therapy to better suppress CRPC in patients that already developed docetaxel resistance.

Cdc20/p55 mediates the resistance to docetaxel in castration-resistant prostate cancer in a Bim-dependent manner.

At least to date, no effective treatment for advanced castration-resistant prostate cancer (CRPC) has been established. Recent studies indicated that cell division cycle 20 homolog (Cdc20) overexpression is associated with poor prognosis in patients with castration-resistant prostate cancer. However, the mechanism of Cdc20 in the development of docetaxel resistance in CRPC remains elusive. In this study, the transcription of Cdc20 was confirmed in three independent CRPC cell lines derived from different tissues, including LNCaP, PC3, and DU145. Docetaxel resistant (DR) cell lines were generated within the background of DU145 and PC3. The protein levels of Cdc20 and the biological phenotype were detected in both wild-type and DR cell lines. To further explore the mechanism of Cdc20 overexpression, stable cell lines with Cdc20 or Bcl-2 interacting mediator of cell death (Bim) deprivation were generated and examined for biological parameters. In addition, a specific Cdc20 inhibitor was used in DR cell lines to explore the potential solution for docetaxel resistant CRPC. Here, we identified Cdc20 is overexpressed in docetaxel resistant CRPC cell lines, including LNCaP, PC3, and DU145. We also reported that DR cell lines, which mimic the recurrent prostate cancer cells after docetaxel treatment, have higher levels of Cdc20 protein compared with the CRPC cell lines. Interestingly, the protein levels of Bim, an E3 ligase substrate of Cdc20, were decreased in DR cell lines compared with the wild-type, while the mRNA levels were similar. More importantly, in DR cell lines, the biological phenotype induced by Cdc20 deletion could be significantly reversed by the additional knockdown of Bim. As a result, docetaxel resistant prostate cancer cells treated with the pharmacological Cdc20 inhibitor became sensitive to docetaxel treatment. In conclusion, our data collectively demonstrated that Cdc20 overexpression facilitates the docetaxel resistant of the CRPC cell lines in a Bim-dependent manner. Furthermore, additionally targeting Cdc20 might be a promising solution for the treatment of the CRPC with docetaxel resistance.

Roles of aldo-keto reductases 1B10 and 1C3 and ATP-binding cassette transporter in docetaxel tolerance

Docetaxel (DTX) is widely used for treatment of inveterate lung and prostate cancers, but its continuous administration elicits the hyposensitivity. Here, we established the DTX-resistant variants of human lung cancer A549 and androgen-independent prostate cancer Du145 cells and found that the resistance development provoked aberrant up-regulations of aldo-keto reductase (AKR) 1B10 and AKR1C3 in A549 and Du145 cells, respectively. In addition, the sensitivity to the DTX toxicity was significantly decreased and increased by overexpression and knockdown of the two AKR isoforms, respectively. Furthermore, the resistant cells exhibited a decreased level of reactive 4-hydroxy-2-nonenal formed during DTX treatment, and the decrease was alleviated by adding the AKR inhibitors, inferring that the two AKRs confer the chemoresistance through elevating the antioxidant properties. The development of DTX resistance was also associated with enhanced expression of an ATP-binding cassette (ABC) transporter ABCB1 among the ABC transporter isoforms. The combined treatment with inhibitors of the two AKRs and ABCB1 additively sensitized the resistant cells to DTX. Intriguingly, the AKR1B10 inhibitor also suppressed the lung cancer cross-resistance against cisplatin. The results suggest that combined treatment with AKRs (1B10 and 1C3) and ABCB1 inhibitors exerts overcoming effect against the cancer resistance to DTX and cisplatin, and can be used as the adjuvant therapy.

Abstract 2142: Plumbagin, a medicinal plant-derived naphthoquinone, inhibits the growth of docetaxel resistant prostate cancer cells via inhibition of the expression of P-glycoprotein and NF-kB activation

Abstract Prostate cancer (PCa) is one of the leading causes of death in men across the world. Despite the initial success of androgen ablation therapy, resistance to anti-androgen therapy manifests by progression to castration resistant prostate cancer (CRPC), which is the end stage that accounts for the majority of PCa patient deaths. Docetaxel (DTX) is an approved drug for the treatment of CRPC. However, development of DTX resistance and toxicity are the major limitations in the use of DTX in the treatment of CRPC. Now we investigated to determine whether these limitations may be overcome by combining DTX with plumbagin (PL). PL is a quinoid constituent isolated from the root of Plumbago zeylanica L. The roots of Plumbago zeylanica L. have been used in Indian medicine for more than 2500 years for treatment of various ailments. We were the first to report that PL inhibits the growth and PCa metastasis. Now we present that PL: 1) sensitizes CRPC cells to DTX and thus reduces its effective therapeutic dose to prevent DTX resistance and toxicity, and 2) inhibits the viability and growth of DTX resistant cells (22Rv1R). In combination therapeutic experiments, the effects of different concentrations of PL (0, 1.0, 2.0, and 5.0 μM) alone or in combination with DTX (0.25 nM and 0.5 nM) on colony formation in 22Rv1 and C4-2 prostate cancer cells, was determined. Combined treatment of PL and DTX has more than additive inhibitory effects on colony formation. This synergism of PL and DTX on colony formation can be explained on the basis of unique mechanism of action of PL and DTX. PL-induced inhibition of PCa growth and metastasis involves inhibition of the expression of multiple targets including PKCϵ, which correlates with the aggressiveness of PCa and plays roles in both androgen independent androgen receptor (AR) activation and promotion of PCa cell survival. DTX targets microtubules, impairs AR signaling, and inhibits cell division. To determine the effects of PL on DTX resistant PCa cells, we established DTX-resistant 22Rv1 cell line (22Rv1R). 22Rv1R overexpresses multidrug resistant protein 1 (MDR1, also known as P-glycoprotein). MDR1 is overexpressed in many tumors and thus causing intrinsic drug resistant. Transcription factor NF-KB, which is linked to cell proliferation and survival, is also activated in 22Rv1R. PL inhibited the colony forming efficiency of 22Rv1R cells which accompanied inhibition of MDR1 expression and NF-KB activation. In summary, the results presented here and the ones reported by us previously with preclinical mouse models (TRAMP and PTEN knockout mice) of PCa indicate that PL has the potential to prevent as well as treat PCa. PL is a potential natural agent worthy of investigation for prevention and treatment of human PCa. (Support: NIH grant CA102431). Citation Format: Ashok Singh, Anupama Singh, Satyamedha Bathula, Saivenkateshkomal Bathula, Ajit K. Verma. Plumbagin, a medicinal plant-derived naphthoquinone, inhibits the growth of docetaxel resistant prostate cancer cells via inhibition of the expression of P-glycoprotein and NF-kB activation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2142.

Abstract 5043: Molecular mechanism of resveratrol induced cell death in multidrug resistance prostate cancer

Abstract In order to understanding the molecular mechanisms of drug resistance as well as enhancing the efficacy of docetaxel we established docetaxel-resistant prostate cancer (PCa) cells. The IC50 for docetaxel in PCa resistant cells was about 50-fold higher than parental PCa cells. In the current study we used resveratrol (RES), a natural compound with chemo preventive potential to test its ability to enhance the effectiveness of docetaxel on PCa as well as to explore the mechanism of acquired docetaxel resistance. PCa (PC3, C4-2B and DU145) cell lines treated with different dose of RES or DTX alone or in combination followed by analyzed cell proliferation (MTT assay), apoptosis (flow-cytometric analysis of annexin V/propidium iodide-stained cells and western blot methods).The expression of ABC transporter markers in the development of docetaxel resistance was examined using RT-PCR. Our result shows that the expression of 18 ABC transporter genes were down regulated in PCa cells at 48 and 72 hrs in RES treatment group. The up regulations of pro-apoptotic proteins (Bax, Bad, Bim) and down regulation of anti-apoptotic protein (Bcl2, Mcl1) further confirmed the effectiveness of RES or DTX alone or in combination with DTX. In conclusion these studies suggest that RES inhibits ABC gene expression and resensitizes docetaxel resistant PCa cells to DTX treatment with enhancing apoptotic cell death. Citation Format: Santosh K. Singh, Saswati Banerjee, James W. Lillard, Rajesh Singh. Molecular mechanism of resveratrol induced cell death in multidrug resistance prostate cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5043.

MP84-05 THE OESTROGEN RECEPTOR IS SIGNIFICANTLY DYSREGULATED IN A MODEL OF DOCETAXEL RESISTANT PROSTATE CANCER AND TAMOXIFEN REVERSES APOPTOTIC RESISTANCE TO DOCETAXEL IN VITRO.

You have accessJournal of UrologyProstate Cancer: Basic Research & Pathophysiology III1 Apr 2016MP84-05 THE OESTROGEN RECEPTOR IS SIGNIFICANTLY DYSREGULATED IN A MODEL OF DOCETAXEL RESISTANT PROSTATE CANCER AND TAMOXIFEN REVERSES APOPTOTIC RESISTANCE TO DOCETAXEL IN VITRO. Dara Lundon, Maria Prencipe, Amanda O'Neill, Sinead Ahearne, Stephen Madden, Padraig Doolan, and William Watson Dara LundonDara Lundon More articles by this author , Maria PrencipeMaria Prencipe More articles by this author , Amanda O'NeillAmanda O'Neill More articles by this author , Sinead AhearneSinead Ahearne More articles by this author , Stephen MaddenStephen Madden More articles by this author , Padraig DoolanPadraig Doolan More articles by this author , and William WatsonWilliam Watson More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2016.02.2229AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES The armamentarium of agents for the treatment of advanced prostate cancer has been significantly augmented in recent years. However the optimal treatment pathway for each man is still poorly defined. Docetaxel is an important chemotherapeutic drug in the treatment of prostate cancer, being amongst the most effective chemotherapeutic agents for the treatment of metastatic castrate-resistant prostate cancer. Docetaxel-resistance is a major obstacle in such patients. Defining the mechanisms underlying resistance so as to inform treatment selection remains a challenge. Previous experiments and publications in our laboratory have shown complex changes in pro and anti-apoptotic proteins in the development of docetaxel resistance. When targeted individually these proteins do not significantly impact upon the resistant phenotype. Our hypothesis was that targeting the central signalling pathways and transcription factors (TF) which control these could represent a more appropriate mechanism for identifying potential therapeutic targets and prognostic markers. Our aims were to identify potential targets/markers and then to undertake a validation of their functional role in a model of docetaxel resistance. METHODS We have developed a number of doxetaxel resistant sublines in PC-3 cells, and undertaken a transciptomic analysis of these cells by DNA microarray analysis using the Affymetrix Human Gene 1.0 ST Array. Using novel bioinformatic techniques, including correspondence, between-group and co-inertia analyses; transcriptional targets suitable for discerning patients with a phenotype of Docetaxel-resistance were identified. Manipulation with tamoxifen and estradiol, was used to determine functional effects on the apoptotic pathway by propidium iodide DNA staining and flow cytometry. RESULTS The TF ESR1, also known as the oestrogen receptor is amongst those predicted to be responsible for the differential gene expression observed in docetaxel-resistant prostate cancer. We have mapped the cell-processes of this TF and can display how it is intimately implicated in regulating docetaxel resistance in prostate cancer cell lines. We have demonstrated the close interplay of ESR1 on apoptosis, response to stress, chemotaxis and chemosensitivity amongst a myriad of inveigling functions. Manipulation of ESR1 in the PC3 docetaxel-resistant cells resulted in altered apoptotic resistance to docetaxel; whereby tamoxifen in combination with docetaxel resulted in a significant increase in apoptosis (p<0.01), and estradiol attenuated resistance (p<0.05) in this cell line model of advanced prostate cancer. CONCLUSIONS Due to the complexity of changes associated with the development of resistance to docetaxel, manipulating the upstream transcription factors and their networks may represent a more comprehensive therapeutic targeting approach for this advanced form of prostate cancer. We have successfully identified ESR1 as being dysregulated in a model of advanced prostate cancer and commenced validation of its functional role in docetaxel resistance in-vitro. The identification of TFs such as ESR1 could be utilised to help stratify men with prostate cancer, as potential therapeutic targets, prognostic aides and predictors of response to docetaxel or combined therapy in men with significant prostate cancer. © 2016FiguresReferencesRelatedDetails Volume 195Issue 4SApril 2016Page: e1091 Advertisement Copyright & Permissions© 2016MetricsAuthor Information Dara Lundon More articles by this author Maria Prencipe More articles by this author Amanda O'Neill More articles by this author Sinead Ahearne More articles by this author Stephen Madden More articles by this author Padraig Doolan More articles by this author William Watson More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Cytokine profiling of docetaxel-resistant castration-resistant prostate cancer.

Background:Docetaxel improves symptoms and survival in metastatic castration-resistant prostate cancer (CRPC). However, ∼50% of patients are chemoresistant. This study examined whether changes in cytokine levels predict for docetaxel resistance in vitro and in a clinical cohort.Methods:PC3 cells or their docetaxel-resistant subline (PC3Rx) were co-cultured with U937 monocytes, with and without docetaxel treatment, and cytokine levels were measured. The circulating levels of 28 cytokines were measured pre-/post cycle 1 of docetaxel from 55 men with CRPC, and compared with prostate-specific antigen (PSA) response.Results:PC3Rx-U937 co-culture expressed more cytokines, chiefly markers of alternative macrophage differentiation, compared with PC3-U937 co-culture. Docetaxel treatment enhanced cytokine production by PC3Rx-U937 co-culture, while reducing cytokine levels in PC3-U937. In patients, changes in the levels of seven circulating cytokines (macrophage inhibitory cytokine 1 (MIC1), interleukin (IL)-1ra, IL-1β, IL-4, IL-6, IL-12 and IFNγ) after cycle 1 of docetaxel were associated with progressive disease (all P<0.05). The combination of changes in MIC1, IL-4 and IL-6 most strongly predicted PSA response (P=0.002).Conclusions:In vitro studies suggest docetaxel resistance is mediated, at least in part, by cytokines induced by the interaction between the docetaxel-resistant tumour cells and macrophages. Early changes in circulating cytokine levels were associated with docetaxel resistance in CRPC patients. When considered together, these data suggest a significant role for the inflammatory response and macrophages in the development of docetaxel resistance in CRPC.

Inhibition of ABCB1 Expression Overcomes Acquired Docetaxel Resistance in Prostate Cancer

Docetaxel is the first-line standard treatment for castration-resistant prostate cancer. However, relapse eventually occurs due to the development of resistance to docetaxel. To unravel the mechanism of acquired docetaxel resistance, we established docetaxel-resistant prostate cancer cells, TaxR, from castration-resistant C4-2B prostate cancer cells. The IC50 for docetaxel in TaxR cells was about 70-fold higher than parental C4-2B cells. Global gene expression analysis revealed alteration of expression of a total of 1,604 genes, with 52% being upregulated and 48% downregulated. ABCB1, which belongs to the ATP-binding cassette (ABC) transporter family, was identified among the top upregulated genes in TaxR cells. The role of ABCB1 in the development of docetaxel resistance was examined. Knockdown of ABCB1 expression by its specific shRNA or inhibitor resensitized docetaxel-resistant TaxR cells to docetaxel treatment by enhancing apoptotic cell death. Furthermore, we identified that apigenin, a natural product of the flavone family, inhibits ABCB1 expression and resensitizes docetaxel-resistant prostate cancer cells to docetaxel treatment. Collectively, these results suggest that overexpression of ABCB1 mediates acquired docetaxel resistance and targeting ABCB1 expression could be a potential approach to resensitize docetaxel-resistant prostate cancer cells to docetaxel treatment.

The role of macrophages in docetaxel (DTX) resistance in castrate-resistant prostate cancer (CRPC).

e22175 Background: DTX improves symptoms and survival in advanced CRPC, however ~ 50% of patients have chemoresistant disease. Given the toxicity of cytotoxic treatment in this population, there is an urgent need for early chemoresistance markers, a greater understanding of chemoresistance mechanisms and the development of new therapies. This study examined whether early changes in cytokine levels predict chemoresistance and explored the role of macrophages in cytokine generation and chemoresistance in vitro. Methods: 28 cytokines were measured pre/post cycle 1 of chemotherapy from 59 men with metastatic CRPC. Cytokine levels were correlated with PSA response and OS. DTX sensitive PC3 and DTX resistant PC3Rx cells were cultured alone and in coculture with U937 monocytes +/- DTX treatment. Conditioned media (CM) was analysed by a 36 cytokine array panel and by ELISA for MIC1. CM from U937 cells exposed to DTX was used to treat PC3 cells combined with escalating DTX doses. Results: Change in the levels of 10 cytokines over 1 cycle of chemotherapy (MIC-1 p=0.003; IL1ra p=0.004; IL1b p=0.01; IL4 p&lt;0.001; IL5 p=0.041; IL6 p=0.003; IL7 p=0.009; IL8 p=0.017; IL12 p=0.004; IFNg p=0.001) was associated with clinical benefit (PR + SD vs PD). The combination of changes in MIC1, IL4 and IL6 best predicted PSA response (ROC AUC 0.88, 95% CI 0.79-0.97). PC3Rx/U937 coculture expressed more cytokines, chiefly markers of alternative macrophage differentiation, compared to PC3/U937 co-culture (IL10 17-fold; IL27 15-fold). DTX treatment enhanced cytokine production by PC3Rx/U937 coculture while reducing cytokine levels in PC3/U937 CM. The IC70 for DTX in PC3 cells incubated with CM from DTX exposed U937 cells was 10 fold greater than with U937 CM without DTX exposure. Conclusions: Early changes in circulating cytokine levels, especially those involved in macrophage activity, were associated with chemoresistance in men with CRPC. In vitro studies revealed a cytokine-mediated interaction between the macrophages and the DTX resistant cells when treated with DTX that was not apparent in the DTX sensitive cell model. When considered together, these data suggest a significant role for host macrophages in the development of DTX resistance in CRPC.

Epithelial-to-Mesenchymal Transition Leads to Docetaxel Resistance in Prostate Cancer and Is Mediated by Reduced Expression of miR-200c and miR-205

Docetaxel is a standard chemotherapy for patients with metastatic prostate cancer. However, the response is rather limited and not all of the patients benefit from this treatment. To uncover key mechanisms of docetaxel insensitivity in prostate cancer, we have established docetaxel-resistant sublines. In this study, we report that docetaxel-resistant cells underwent an epithelial-to-mesenchymal transition during the selection process, leading to diminished E-cadherin levels and up-regulation of mesenchymal markers. Screening for key regulators of an epithelial phenotype revealed a significantly reduced expression of microRNA (miR)-200c and miR-205 in docetaxel-resistant cells. Transfection of either microRNA (miRNA) resulted in re-expression of E-cadherin. Functional assays confirmed reduced adhesive and increased invasive and migratory abilities. Furthermore, we detected an increased subpopulation with stem cell-like properties in resistant cells. Tissue microarray analysis revealed a reduced E-cadherin expression in tumors after neoadjuvant chemotherapy. Low E-cadherin levels could be linked to tumor relapse. The present study uncovers epithelial-to-mesenchymal transition as a hallmark of docetaxel resistance. Therefore, we suggest that this mechanism is at least in part responsible for chemotherapy failure, with implications for the development of novel therapeutics.

P1-013 - High Expression of Acetyl-Tubulin as a Chemoresistant Biomarker in Human Docetaxel-Resistance Prostate Cancer Cells

ABSTRACT Background Docetaxel-based chemotherapy has generally been considered as one of the effective treatments for prostate cancer. Unfortunately, clinical treatment with docetaxel often encounters a number of undesirable side-effects, including drug resistance. Therefore, it has become essential to identify molecular events that may be associated with the development of docetaxel resistance. Tubulin isoforms have been previously examined for their resistant ability to docetaxel in many cancers, but their real mechanisms remained unclear. In this study, we evaluated the feasibility of employing docetaxel as a cytotoxic agent for PC3 cells and to examine the role of acetyl-tubulin in docetaxel-resistant prostate cancer. Methods A series of docetaxel-resistant PC3/DX cell subclones was established by chronically exposing PC3 to progressively increased concentrations of docetaxel. Herein, we characterized the docetaxel-mediated cytoxicity and molecular events in PC3 and PC3/DX cells by the MTT assay, western blotting, RT–PCR and real-time PCR. Results Our results showed that levels of acetyl-tubulin, a-tubulin, b-tubulin, γ-tubulin and bIII-tubulin were significantly higher expression in PC3/DX than in parental PC3 cells by western blotting analysis. PC/DX with greater resistance to docetaxel had higher levels of acetyl-tubulin than in PC3 cells. The expression of acetyl-tubulin was gradually increased by docetaxel in a dose- and time-dependent manner in PC3 cells. Histone deacetylase 6, a deacetyl enzyme of tubulin, mRNA and protein levels were significantly decreased in PC3/DX than in PC3 cells. Interestingly, we also found an evident up-regulation of acetyl-tubulin protein expression after recombinant epidermal growth factor treatment in a time-dependent manner in PC3 cells. Conclusions Up-regulation of acetyl-tubulin may play an important chemoresistant role in docetaxel-resistant prostate cancer.

Identification of microRNA profiles in docetaxel‐resistant human non‐small cell lung carcinoma cells (SPC‐A1)

Docetaxel has been used as first-line chemotherapy in advanced non-small cell lung carcinoma (NSCLC), but further extensive and effective application is prevented by drug resistance. MicroRNAs (miRNAs) have recently been identified as important posttranscriptional regulators, which are involved in various biological processes. The aim of this study was to identify microRNA expression profiles involved in the development of docetaxel resistance in NSCLC. Here, microarray chip technology was employed to identify miRNA expression profiles in docetaxel-resistant human NSCLC cell line (SPC-A1/docetaxel). Then, the changes of miRNAs expression (>2-fold compared with control SPC-A1 cell line) were testified by quantitative real-time RT-PCR (qRT-PCR) assay. Furthermore, the potential target genes regulated by selected miRNAs were analysed by various target prediction tools. The expression of a total of 52 miRNAs showed significant difference between SPC-A1/docetaxel cells and control SPC-A1 cells (P < 0.01). Six miRNAs (miR-192, 200b, 194, 424, 98 and 212) exhibited more than 2-fold changes in their expression levels, which were validated by qRT-PCR. The expression of three miRNAs (miR-200b, 194 and 212) was significantly down-regulated in SPC-A1/docetaxel cells, while the expression of other three miRNAs (miR-192, 424 and 98) was significantly up-regulated in SPC-A1/docetaxel cells (P < 0.01). Potential target genes controlled by six selected miRNAs were divided into four groups according to various functions: apoptosis and proliferation (71 genes), cell cycle (68 genes), DNA damage (26 genes) and DNA repair (59 genes). The expression of a few target genes in SPC-A1/docetaxel and SPC-A1 cells were further confirmed by qRT-PCR and Western blot. Taken together, the identification of microRNA expression profiles in docetaxel-resistant NSCLC cells could provide a better understanding of mechanisms involved in drug sensitivity or resistance, which would be helpful to develop novel strategies for targeted therapies in chemorefractive NSCLC patients.

Identification of Candidate Biomarkers of Therapeutic Response to Docetaxel by Proteomic Profiling

Docetaxel chemotherapy improves symptoms and survival in men with metastatic hormone-refractory prostate cancer (HRPC). However, approximately 50% of patients do not respond to Docetaxel and are exposed to significant toxicity without direct benefit. This study aimed to identify novel therapeutic targets and predictive biomarkers of Docetaxel resistance in HRPC. We used iTRAQ-mass spectrometry analysis to identify proteins associated with the development of Docetaxel resistance using Docetaxel-sensitive PC3 cells and Docetaxel-resistant PC3-Rx cells developed by Docetaxel dose escalation. Functional validation experiments were performed using recombinant protein treatment and siRNA knockdown experiments. Serum/plasma levels of the targets in patient samples were measured by ELISA. The IC(50) for Docetaxel in the PC3-Rx cells was 13-fold greater than the parent PC-3 cell line (P = 0.004). Protein profiling identified MIC-1 and AGR2 as respectively up-regulated and down-regulated in Docetaxel-resistant cells. PC-3 cells treated with recombinant MIC-1 also became resistant to Docetaxel (P = 0.03). Conversely, treating PC3-Rx cells with MIC-1 siRNA restored sensitivity to Docetaxel (P = 0.02). Knockdown of AGR2 expression in PC3 cells resulted in Docetaxel resistance (P = 0.007). Furthermore, increased serum/plasma levels of MIC-1 after cycle one of chemotherapy were associated with progression of the cancer (P = 0.006) and shorter survival after treatment (P = 0.002). These results suggest that both AGR2 and MIC-1 play a role in Docetaxel resistance in HRPC. In addition, an increase in serum/plasma MIC-1 level after cycle one of Docetaxel may be an indication to abandon further treatment. Further investigation of MIC-1 as a biomarker and therapeutic target for Docetaxel resistance in HRPC is warranted.

Novel role of Stat1 in the development of docetaxel resistance in prostate tumor cells

A major obstacle for clinicians in the treatment of advanced prostate cancer is the inevitable progression to chemoresistance, especially to docetaxel. It is essential to understand the molecular events that lead to docetaxel resistance in order to identify means to prevent or interfere with chemoresistance. In initial attempts to detect these events, we analysed genomic differences between non-resistant and docetaxel-resistant prostate tumor cells and, of the genes modulated by docetaxel treatment, we observed Stat1 and clusterin gene expression heightened in the resistant phenotype. In this study, we provide biochemical and biological evidence that these two gene products are related. Stat1 and clusterin protein expression was induced upon docetaxel treatment of DU145 cells and highly overexpressed in the docetaxel-resistant DU145 cells (DU145-DR). The increase in total Stat1 corresponded to an increase in phosphorylated Stat1. Interestingly, there was no detectable difference between DU145 and DU145-DR cells expression of total Stat3 and phosphorylated Stat3. Treatment of DU145-DR cells with small interfering RNA targeted for Stat1 not only resulted in the knockdown of Stat1 expression, but it also caused the inhibition of clusterin expression. Thus, Stat1 appears to play a key role in the regulation of clusterin. Remarkably, inhibition of Stat1 or clusterin expression resulted in the re-sensitization of DU145-DR cells to docetaxel. These results offer the first evidence that Stat1, and its subsequent regulation of clusterin, are essential for docetaxel resistance in prostate cancer. Targeting this pathway could be a potential therapeutic means for intervention of docetaxel resistance.