Docetaxel-resistant Castration-resistant Prostate Cancer Research Articles

β-Arrestin2 promotes docetaxel resistance of castration-resistant prostate cancer via promoting hnRNP A1-mediated PKM2 alternative splicing

PurposeTo investigate the influence of β-arrestin2 on the docetaxel resistance in castration-resistant prostate cancer (CRPC) and elucidate the underlying molecular mechanisms.MethodsPC3 and DU145 cells with stable β-arrestin2 overexpression and C4-2 cells with stable β-arrestin2 knockdown, were constructed via using lentivirus and puromycin selection. MTT and colony formation assays were carried out to investigate the effect of β-arrestin2 expression on the docetaxel resistance of CRPC cells. Glycolysis analysis was used to assess the glycolytic capacity modulated by β-arrestin2. GO enrichment analysis, gene set enrichment analysis and Spearman correlation test were carried out to explore the potential biological function and mechanism via using public data from GEO and TCGA. The expressions of PKM2, Phospho-PKM2, Phospho-ERK1/2 and hnRNP A1 were detected by western blot. Functional blocking experiments were carried out to confirm the roles of PKM2 and hnRNP A1 in the regulation of β-arrestin2’s biological functions via silencing PKM2 or hnRNP A1 expression in cells with stable β-arrestin2 overexpression. Finally, nude mice xenograft models were established to confirm the experimental results of cell experiments.Resultsβ-Arrestin2 significantly decreased the sensitivity of CRPC cells to docetaxel stimulation, through enhancing the phosphorylation and expression of PKM2. Additionally, β-arrestin2 increased PKM2 phosphorylation via the ERK1/2 signaling pathway and induced PKM2 expression in a post-transcriptional manner through an hnRNP A1-dependent PKM alternative splicing mechanism, rather than by inhibiting its ubiquitination degradation.ConclusionOur findings indicate that the β-arrestin2/hnRNP A1/PKM2 pathway could be a promising target for treating docetaxel-resistant CRPC.

Inhibition of CHRM1 reverts docetaxel resistance in castration‐resistant prostate cancer

BackgroundChemotherapy remains the major treatment option for castration‐resistant prostate cancer (CRPC). Docetaxel (DTX)‐based chemotherapy provides significant survival benefit in patients with CRPC. However, the clinical utility of DTX is compromised when resistance is encountered. Therefore, a more thorough understanding of mechanisms underlying DTX resistance may potentially improve survival in patients with CRPC. The objective of this study is to investigate the role and mechanism of cholinergic receptor muscarinic 1 (CHRM1) activation upon chemotherapy resistance in PC and the efficacy of the CHRM1 antagonist dicyclomine (Dic) in treating DTX‐resistant PC. The hypothesis is that CHRM1 is essential for developing chemotherapy resistance in PC cells and driving survival and growth of DTX‐resistant PC cells.MethodsThe CHRM1 expression levels and their association with DTX resistance were determined in PC clinical specimens and cultured cells. Human PC cells subjected to stable knockdown or overexpression of CHRM1 were evaluated for their response to DTX by cell proliferation, colony and tumorsphere formation, and TUNEL assays. Synergistic growth inhibition by combination treatment with DTX and Dic was assessed by cell viability and tumorgenicity assays.ResultsCHRM1 expression was elevated under DTX treatment in CRPC 22Rv1 and DU145 cells, which was paralleled by higher CHRM1 expression in these cells after acquiring resistance to DTX (22Rv1DTXR and DU145DTXR). Immunohistochemical analyses revealed higher CHRM1 protein levels in longitudinally collected tumor tissues post‐ versus pre‐chemotherapy from a PC patient cohort. Enforced expression of CHRM1 increased DTX IC50 by up to 5‐fold, along with reduced cell apoptosis marker c‐PARP1 expression, in 22Rv1 and DU145 cells. Conversely, silencing of CHRM1 in 22Rv1DTXR cells decreased cell proliferation, invasion, and colony and tumorsphere formation. CHRM1 inactivation with Dic in combination with DTX resulted in more cell death of both DTX‐sensitive (22Rv1 and DU145) and ‐resistant (22Rv1DTXR, DU145DTXR and PC‐3DTXR) cells compared to either treatment alone. Further, Dic restored DTX sensitivity to suppress the growth of DTX‐resistant cells and xenograft tumors (22Rv1DTXR and PC‐3DTXR). Mechanistically, CHRM1 activation upregulated phosphoprotein levels of ERK, JNK and p38, which all are key players in the MAPK pathway and potential mediators driving chemoresistance in cancer cells, while Dic treatment reduced their phosphorylation levels in 22Rv1DTXR and DU145DTXR cells.ConclusionOur results suggest that CHRM1 contributes to the development of DTX resistance in CRPC, which can be reversed by genetic or pharmacologic inactivation of CHRM1, providing new insights into developing a CHRM1‐targeted therapeutic strategy for overcoming DTX‐based chemotherapy resistance in CRPC.

Lipin Expression Correlates to Differences in Phospholipid Profiles in Drug‐Resistant Prostate Cancer Cells

PURPOSEThe association between clinical outcomes of prostate cancer progression and lipid remodeling is not well understood. Although it is known that increases in select circulating lipids correlate to decreased patient survival, the mechanisms mediating alterations in these lipids are not fully explained. We addressed this gap‐in‐knowledge using in vitro models of non‐cancerous, hormone‐sensitive, castration‐resistant prostate cancer (CRPC), and docetaxel resistant (DR) CRPC cell lines combined with quantitative HPLC‐ESI‐Orbitrap‐MS lipidomic analysis.HYPOTHESISWe hypothesized that the expression of lipin (phosphatidic acid phosphatase), an enzyme mediating the conversion of phosphatidic acid to diacylglycerol, correlates to changes in lipidomic profiles in prostate cancer cells. We further hypothesized that the lipidomic profile of metastatic CRPC cell lines (PC‐3 and DU‐145) will shift as they are undergoing multiple treatments of docetaxel.METHODSLipids were isolated using the Bligh‐Dyer extraction from non‐cancerous, hormone‐sensitive, CRPC, and DR‐CRPC cell lines. Lipids were initially analyzed using an untargeted shotgun approach (ESI‐MS), followed by a targeted‐based quantitative HPLC‐ESI‐Orbitrap‐MS approach. Post data acquisition were based on multiple methods including isotope, carbon number (to internal standards) and ionization efficiency‐based corrections. Online resources and software's utilized included MetaboAnalyst, LIPIDMATCH, LIPID MAPS, XCMS, and MZmine.Lipin‐1 expression in cells was determined using immunoblot analysis. Docetaxel‐sensitive traits were obtained after five treatments of 1 nM docetaxel between passages of the CRPC cell line PC‐3. Results were demonstrated by a 3‐(4,5‐Dimethylthiazol‐2‐yl)‐2,5‐Diphenyltetrazolium Bromide (MTT) assay.RESULTSOur data showed significant changes in the abundance of lipid species between the various cell lines and a correlation between select lipid species and the progression of prostate cancer. Prominent among these were the levels of phosphatidic acid (PA) and diacylglycerol (DAG). Most notably, PA levels in cells tended to inversely correlate with DAG levels. These levels correlated to the decreased expression on lipin, specifically lipin‐1.CONCLUSIONSDistinct phospholipid classes were observed to have increased levels in prostate cancer cells, as compared to non‐cancer cells. The protein expression of lipin‐1 in most prostate cell lines correlated to the abundance of PA, excluding the DR cell lines. This suggests that altered lipin expression correlates to decreased survival. Additionally, dosing the parent and treated PC‐3 cells with a range of docetaxel indicated that the 5x1nM treated cells had a larger cytotoxic effect to docetaxel, as compared to the non‐treated cells.REFERENCESIngram, L.M., et al., Identification of lipidomic profiles associated with drug‐resistant prostate cancer cells. Lipids Health Dis, 2021. 20(1): p. 15.

Topoisomerase II alpha inhibition can overcome taxane-resistant prostate cancer through DNA repair pathways

Cabazitaxel (CBZ) is approved for the treatment of docetaxel-resistant castration-resistant prostate cancer (CRPC). However, its efficacy against CRPC is limited, and there are no effective treatments for CBZ-resistant CRPC. This study explored the optimal treatment for CRPC in the post-cabazitaxel setting. PC3 (CBZ-sensitive) and PC3CR cells (CBZ-resistant) were used in this study. We performed in silico drug screening for candidate drugs that could reprogram the gene expression signature of PC3CR cells. The in vivo effect of the drug combination was tested in xenograft mice models. We identified etoposide (VP16) as a promising treatment candidate for CBZ-resistant CRPC. The WST assay revealed that VP16 had a significant antitumor effect on PC3CR cells. PC3CR cells exhibited significantly higher topoisomerase II alpha (TOP2A) expression than PC3 cells. Higher TOP2A expression was a poor prognostic factor in The Cancer Genome Atlas prostate cancer cohort. In the Fred Hutchinson Cancer Research Center dataset, docetaxel-exposed tissues and metastatic tumors had higher TOP2A expression. In addition, VP16 significantly inhibited the growth of tumors generated from both cell lines. Based on these findings, VP16-based chemotherapy may be an optimal treatment for CPRC in the post-CBZ setting.

Androgen receptor negatively regulates mitotic checkpoint signaling to induce docetaxel resistance in castration-resistant prostate cancer.

BackgroundDespite multiple treatment advances for castration‐resistant prostate cancer (CRPC), there are currently no curative therapies and patients ultimately to succumb to the disease. Docetaxel (DTX) is the standard first‐line chemotherapy for patients with metastatic CRPC; however, drug resistance is inevitable and often develops rapidly, leading to disease progression in nearly all patients. In contrast, when DTX is deployed with androgen deprivation therapy in castration‐sensitive disease, more durable responses and improved outcomes are observed, suggesting that aberrant androgen receptor (AR) signaling accelerates DTX resistance in CRPC. In this study, we demonstrate that AR dysregulates the mitotic checkpoint, a critical pathway involved in the anticancer action of DTX.MethodsAndrogen‐dependent and independent cell lines were used to evaluate the role of AR in DTX resistance. Impact of drug treatment on cell viability, survival, and cell‐cycle distribution were determined by plate‐based viability assay, clonogenic assay, and cell‐cycle analysis by flow cytometry, respectively. Mitotic checkpoint kinase signal transduction and apoptosis activation was evaluated by Western blotting. Pathway gene expression analysis was evaluated by RT‐PCR. A Bliss independence model was used to calculate synergy scores for drug combination studies.ResultsActivation of AR in hormone‐sensitive cells induces a rescue phenotype by increasing cell viability and survival and attenuating G2/M arrest in response to DTX. Analysis of mitotic checkpoint signaling shows that AR negatively regulates spindle checkpoint signaling, resulting in premature mitotic progression and evasion of apoptosis. This phenotype is characteristic of mitotic slippage and is also observed in CRPC cell lines where we demonstrate involvement of AR splice variant AR‐v7 in dysregulation of checkpoint signaling. Our findings suggest that DTX resistance is mediated through mechanisms that drive premature mitotic exit. Using pharmacologic inhibitors of anaphase‐promoting complex/cyclosome and polo‐like kinase 1, we show that blocking mitotic exit induces mitotic arrest, apoptosis, and synergistically inhibits cell survival in combination with DTX.ConclusionOur results suggest that targeting the mechanisms of dysregulated mitotic checkpoint signaling in AR‐reactivated tumors has significant clinical potential to extend treatment benefit with DTX and improve outcomes in patients with lethal prostate cancer.

Zhoushi Qi Ling decoction represses docetaxel resistance and glycolysis of castration-resistant prostate cancer via regulation of SNHG10/miR-1271-5p/TRIM66 axis

Docetaxel resistance developed in half of castration-resistant prostate cancer (CRPC) patients hinders its long-term clinical application. The current study was designed to investigate the effects of Chinese medicine Zhoushi Qi Ling decoction on the docetaxel resistance of prostate cancer as well as elucidate the underlying molecular mechanism. In our study, Qi Ling significantly decreased viability and colony formation as well as increased apoptosis of docetaxel-resistant (DR) CRPC cells. Qi Ling-treated DR cells exhibited decreased glucose consumption, lactate release and pyruvate production. Moreover, lncRNA SNHG10 was upregulated in DR tissues of CRPC patients and was negatively correlated with the progression-free survival. Bioinformatics analysis indicated miR-1271-5p as the associated miRNA possibly binding with SNHG10. miR-1271-5p up-regulation dramatically decreased the luciferase activity of SNHG10 in DR cells. SNHG10 knockdown sharply increased the expression of miR1271-5p in DR cells. Targetscan predicted TRIM66 as one of the downstream targets of miR-1271-5p. miR-1271-5p up-regulation drastically reduced luciferase activity as well as TRIM66 expression in DR cells. Also, the knockdown of SNHG10 remarkably suppressed the expression of TRIM66 in DR cells. Additionally, Qi Ling treatment reduced SNHG10 and TRIM66, while increased miR1271-5p, in DR cells. In summary, Qi Ling inhibited docetaxel resistance and glycolysis of CRPC possibly via SNHG10/miR-1271-5p/TRIM66 pathway.

Discovery of a new candidate drug to overcome cabazitaxel-resistant gene signature in castration-resistant prostate cancer by in silico screening

BackgroundThe taxane cabazitaxel (CBZ) is a promising treatment for docetaxel-resistant castration-resistant prostate cancer (CRPC). However, the survival benefit with CBZ for patients with CRPC is limited. This study used screening tests for candidate drugs targeting CBZ-resistant-related gene expression and identified pimozide as a potential candidate for overcoming CBZ resistance in CRPC.MethodsWe established CBZ-resistant cell lines, DU145CR and PC3CR by incubating DU145 cells and PC3 cells with gradually increasing concentrations of CBZ. We performed in silico drug screening for candidate drugs that could reprogram the gene expression signature of a CBZ-resistant prostate cancer cells using a Connectivity Map. The in vivo effect of the drug combination was tested in xenograft mice models.ResultsWe identified pimozide as a promising candidate drug for CBZ-resistant CRPC. Pimozide had a significant antitumor effect on DU145CR cells. Moreover, combination treatment with pimozide and CBZ had a synergic effect for DU145CR cells in vitro and in vivo. Microarray analysis identified AURKB and KIF20A as potential targets of pimozide in CBZ-resistant CRPC. DU145CR had significantly higher AURKB and KIF20A expression compared with a non-CBZ-resistant cell line. Inhibition of AURKB and KIF20A had an antitumor effect in DU145CR xenograft tumors. Higher expression of AURKB and KIF20A was a poor prognostic factor of TGCA prostate cancer cohort. CBZ-resistant prostate cancer tissues in our institution had higher AURKB and KIF20A expression.ConclusionsPimozide appears to be a promising drug to overcome CBZ resistance in CRPC by targeting AURKB and KIF20A.

Molecular Mechanism Mediating Cytotoxic Activity of Cabazitaxel in Docetaxel-resistant Human Prostate Cancer Cells.

Cabazitaxel is known to be effective in patients with castration-resistant prostate cancer (CRPC) showing resistance to docetaxel. The objective of this study was to investigate the molecular mechanism mediating cytotoxic activity of cabazitaxel in docetaxel-resistant human CRPC cells. Parental human CRPC cell line PC3 (PC3/P) was continuously exposed to increasing doses of docetaxel, and a cell line resistant to docetaxel, PC3/R, was developed. Phenotypic differences between these cell lines were investigated. There were no significant differences in sensitivity to cabazitaxel between PC3/P and PC3/R. In PC3/P, both docetaxel and cabazitaxel markedly inhibited the phosphorylation of AKT serine/threonine kinase 1 (AKT) and p44/42 mitogen-activated protein kinase (MAPK). In PC3/R, however, phosphorylation of AKT and p44/42 MAPK were maintained following treatment with docetaxel, whereas treatment with cabazitaxel resulted in the marked down-regulation of phosphorylation of AKT but not that of p44/42 MAPK. Furthermore, additional treatment of PC3/R with a specific inhibitor of AKT significantly enhanced the cytotoxic activity of docetaxel but not that of cabazitaxel. Growth of PC3/R in nude mice after treatment with cabazitaxel was significantly inhibited compared with that after treatment with docetaxel. Antitumor activity of cabazitaxel in docetaxel-resistant CRPC cells was explained, at least in part, by the inactivation of persistently phosphorylated AKT even after treatment with docetaxel.

A Long Noncoding RNA, GAS5 Can Be a Biomarker for Docetaxel Response in Castration Resistant Prostate Cancer.

While functional studies of long noncoding RNAs (lncRNAs) have mostly focused on how they influence disease diagnosis and prognosis, the pharmacogenomic relevance of lncRNAs remains largely unknown. Here, we test the hypothesis that the expression of a lncRNA, grow arrest-specific 5 (GAS5) can be a biomarker for docetaxel response in castration resistant prostate cancer (CRPC) using both prostate cancer (PCa) cell lines and CRPC patient datasets. Our results suggest that lower GAS5 expression is associated with docetaxel resistance in both PCa cell lines and CRPC patients. Further experiments also suggest that GAS5 is downregulated in docetaxel resistant CRPC cell lines, which reinforces its potential as a biomarker for docetaxel response. To examine the underlying biological mechanisms, we transiently knockdown GAS5 expression in PCa cell lines and then subject the cells to docetaxel treatment overtime. We did not observe a decrease in docetaxel induced growth inhibition or apoptosis in the siRNA treated cells. The findings suggest that there is no direct causal relationship between change in GAS5 expression and docetaxel response. Subsequently, we explored the indirect regulation among GAS5, ATP binding cassette subfamily B member 1 (ABCB1), and docetaxel sensitivity. We showed that transient knockdown GAS5 did not lead to significant changes in ABCB1 expression. Therefore, we rule out the hypothesis that GAS5 directly down regulate ABCB1 that lead to docetaxel sensitivity. In conclusion, our work suggests that GAS5 can serve as a predictive biomarker for docetaxel response in CRPC; however, the exact mechanism behind the observed correlation remain to be elucidated.

Potent antitumour of the mTORC1/2 dual inhibitor AZD2014 in docetaxel-sensitive and docetaxel-resistant castration-resistant prostate cancer cells.

Recent studies indicate mammalian target of rapamycin (mTOR) may play an important role in PCa progression and drug resistance. Here, we investigated the effects of a novel mTORC1/C2 dual inhibitor, AZD2014, on naive and docetaxel (Doc)‐pre‐treated castration‐resistant PCa (CRPC) cells and explored its therapeutic potential in CRPCs. In the current study, AZD2014 has a greater inhibitory effect against 4EBP1 and AKT phosphorylation than rapamycin in CRPC cells and prevented the feedback activation of AKT signalling. Importantly, AZD2014 suppressed CRPC cell growth in vitro by suppressing proliferation, apoptosis, cell cycle arrest at G1 phase and autophagy to a greater extent than rapamycin. Moreover, AZD2014 was more efficacious than rapamycin in inhibiting migration, invasion and EMT progression in Doc‐sensitive and Doc‐resistant CRPC cells. Overall, AZD2014 showed significant antitumour effects. Thereby, the current study highlights a reliable theoretical basis for the clinical application of AZD2014 in both Doc‐sensitive and Doc‐resistant CRPCs.

Curcumin Nanoparticles and Their Cytotoxicity in Docetaxel-Resistant Castration-Resistant Prostate Cancer Cells.

Most prostate cancer patients develop resistance to anti-androgen therapy. This is referred to as castration-resistant prostate cancer (CRPC). Docetaxel (DTX) is the mainstay treatment against CRPC. However, over time patients eventually develop DTX resistance, which is the cause of the cancer-related mortality. Curcumin (CUR) as a natural compound has been shown to have very broad pharmacological activities, e.g., anti-inflammatory and antioxidant properties. However, CUR is very hydrophobic. The objective of this study was to develop CUR nanoparticles (NPs) and evaluate their cytotoxicity in DTX-resistant CRPC cells for the treatment of DTX-resistant CRPC. We tested solubility of CUR in different lipids and surfactants. Finally, Miglyol 812 and D-alpha-tocopheryl poly (ethylene glycol) succinate 1000 (TPGS) were chosen to prepare lipid-based NPs for CUR. We fully characterized CUR NPs that had particle size < 150 nm, high drug loading (7.5%), and entrapment efficiency (90%). Moreover, the CUR NPs were successfully lyophilized without using cryoprotectants. We tested the cytotoxicity of blank NPs, free CUR, and CUR NPs in sensitive DU145 and PC3 cells as well as their matching docetaxel-resistant cells. Cytotoxicity studies showed that blank NPs were very safe for all tested prostate cancer cell lines. Free CUR overcame the resistance in PC3 cells, but not in DU145 cells. In contrast, CUR NPs significantly increased CUR potency in all tested cell lines. Importantly, CUR NPs completely restored CUR potency in both resistant DU145 and PC3 cells. These results demonstrate that the CUR NPs have potential to overcome DTX resistance in CRPC.

CD44v8-10 mRNA contained in serum exosomes as a diagnostic marker for docetaxel resistance in prostate cancer patients

BackgroundDocetaxel is first-line chemotherapy for castration-resistant prostate cancer (CRPC), but most patients acquire docetaxel resistance. CD44 has been shown to be involved in drug resistance of cancers including prostate cancer. We hypothesized that CD44 in serum exosomes could be a diagnostic marker for docetaxel resistance in CRPC patients. In this study, we examined CD44 protein and mRNA expression in cell lysates and exosomes isolated from prostate cancer cells, evaluated the effect of CD44v8-10 knockdown on docetaxel sensitivity and measured CD44 mRNA copy numbers contained in serum exosomes in prostate cancer patients. Materials and methodsDocetaxel-sensitive PC-3 prostate cancer cells and docetaxel-resistant PC-3R cells established previously from parental PC-3 cells were used. CD44v8-10 knockdown was performed by siRNA transfection. Blood was collected from 50 docetaxel-naïve and 10 docetaxel-resistant patients and 15 control males. CD44 protein expression was evaluated by Western blotting. CD44 mRNA expression was measured by RT-digital PCR. ResultsThe levels of CD44v8-10 protein and mRNA in cell lysates and exosomes were higher in PC-3R cells than in PC-3 cells. CD44v8-10 knockdown significantly increased docetaxel sensitivity of PC-3R cells. The CD44v8-10 mRNA copy numbers in serum exosomes were higher in docetaxel-resistant patients than in docetaxel-naïve patients and control males (median 46, 12 and 17 copies/mL serum, respectively, P = 0.032). In contrast, the serum exosomal mRNA copy numbers of CD44 standard isoform (CD44s) were not different among 3 groups (median 25, 14 and 13 copies/mL serum, respectively, P = 0.150). ConclusionsCD44v8-10 may be involved in docetaxel resistance in prostate cancer and serum exosomal CD44v8-10 mRNA could be a diagnostic marker for docetaxel-resistant CRPC.

Lipidomic Analysis Demonstrates that 36:1 Phosphatidylcholine is Enriched in Cell Models of Drug Resistance Prostate Cancer

A lack of understanding exists between the association between circulating lipids and clinical outcomes of drug‐resistant castration‐resistant prostate cancer (CRPC). Although an increase in select circulating lipids correlates to decreased patient survival, neither the mechanisms mediating alterations in these lipids, nor the correlation to drug resistance are well characterized. This gap‐in‐knowledge was addressed using in vitro models of non‐cancerous, hormone‐sensitive, CRPC and drug‐resistant cell lines combined with quantitative HPLC‐ESI‐Orbitrap‐MS lipidomic analysis.Lipid extracts were obtained using Bligh‐Dyer (methanol/chloroform/water) extraction from non‐cancerous, hormone‐sensitive, CRPC and drug‐resistant prostate cell lines. Lipids present in both cells and media were initially analyzed using an untargeted shotgun approach (ESI‐MS), followed by a targeted‐based quantitative HPLC‐ESI‐Orbitrap‐MS approach. Shotgun experiments were performed using commercially available standards. For target‐based lipidomics, LC separation yielded specific lipid class separation prior to ESI/MS/MS enabling enhanced detection of lipids suppressed in the shotgun approach. Post data acquisition for both shotgun and HPLC‐ESI/MS/MS lipidomics were based on multiple methods including isotope, carbon number (to internal standards) and ionization efficiency‐based corrections. Pathway analysis was also performed on the various prostate cell lines. Online resources and software’s utilized included MetaboAnalyst, LIPIDMATCH LIPID MAPS, XCMS, and MZmine.Principle component analysis (PCA) generated scores plots of non‐cancerous, hormone‐sensitive, CRPC parent cell lines and drug‐resistant CRPC cell lines in the positive mode (ESI+) had a total variance of 66%. Additionally, a distinct separation between drug‐resistant and parent control cell lines was shown. Several discriminatory ion features were identified between prostate cell lines, especially between drug‐resistant and parent control neuroendocrine cell lines. Of these discriminatory features, 83 showed significant alterations. Two of the identified lipids had alterations matching those measured in plasma from prostate cancer patients.Distinct phospholipid classes were observed to have increased levels in prostate cancer cells, when compared to non‐cancer cells. Lipidomic analysis identified phosphatidylcholines (PC 36:1), whose levels were increased in Docetaxel resistant CRPC. These data also show that the lipidomic profiles of prostate cancer cell lines mirror that seen in the plasma of prostate cancer patients. These data may lead to new early detection strategies and more personalized treatment options for patients.Support or Funding InformationDepartment of Defense Prostate Cancer Research Program Idea Development Award (PC150431 GRANT11996600)Phospholipids in Prostate Cancer(A. Scores Plot) PCA scores plot comparing drug‐resistant CRPC cells to DU145 control parent cells in the positive mode (ESI+). ~80% of the total variance is explained by Component 1 and Component 2. (B. MTT Assay) MTT assay shows decrease in MTT staining in parent cell lines as compared to docetaxel‐resistant cell lines. This suggests DU145‐DR (DR) maintain resistance to Docetaxel as compared to DU145 parent cell line. (C. Heat Map) Each row represents a metabolite feature and each column represents a sample. Metabolite features whose levels vary significantly (p&lt; 0.01) are projected on the heat map and used for sample clustering displayed above. (D. Cloud Plot) Differential feature plot: Only features that are dysregulated in both DU145‐DR cells and DU145‐DR media (p‐value&lt;= 0.05 threshold, fold change &gt;= 1.5 threshold) are displayed above. Up‐regulated features (features that have a positive fold change) are graphed above the x‐axis while down‐regulated features (features that have a negative fold change) are graphed below the x‐axis.Figure 1PC 36‐1 in Prostate Cancer(A) Cloud plot showing 7460 discriminatory ion features (positive ion data) in human cell lines. Only dysregulated features are displayed above (p‐value &lt;= 0.05 threshold, fold change &gt;= 1.5 threshold). The radius of each bubble corresponds to the log fold change of that feature. Features with higher fold change (or higher intensity) will have larger radii. The shade of bubbles corresponds to the magnitude of the p‐value (the darker the color, the smaller the p‐value). The statistical significance of the fold change, as calculated by a Welch t‐test with unequal variances.Figure 6. (B) Box‐plot identified 788.6 feature in human cell lines. Of the lipids identified by the cloud plot, PC(36:1) was one of the few lipid species enriched in all drug resistant prostate cancer cell lines as compared to control cells. This was previously observed in castration‐resistant prostate cancer patient‐derived plasma samples.Figure 2

Docetaxel-resistant prostate cancer cells become sensitive to gemcitabine due to the upregulation of ABCB1.

Docetaxel is the preferred chemotherapeutic agent for hormone-refractory prostate cancer (PC) patients. However, patients eventually develop docetaxel resistance, and no effective treatment options are available for them. We aimed to establish docetaxel resistance in castration-resistant prostate cancer (CRPC) cell lines (DU145/TXR, PC-3/TXR, and CWR22/TXR) and characterized transcriptional changes upon acquiring resistance to the docetaxel. Human PC cells (DU145, PC-3, CWR22) and all docetaxel-resistant cells were maintained in Roswell Park Memorial Institute Medium (RPMI) 1640 media supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. ABCB1 was detected by using both parental and docetaxel-resistant CRPCs prepared for flow cytometry. For the evaluation of tumor-suppressive effects under each chemotherapeutic agent, subcutaneous xenografts of DU145 or DU145/TXR were implanted at the mouse flank. The P-glycoprotein-encoding gene ABCB1 was distinctively upregulated in the resistant cells, and its overexpression played an essential role in docetaxel resistance in CRPC. When tested for the cytotoxicity of gemcitabine, another option for chemotherapy, the docetaxel-resistant cells were shown to become sensitive to the drug, implying additional phenotypic transformation in the docetaxel-resistant cells. Studies using xenograft animal models demonstrated that the growth of tumors composed of both docetaxel-sensitive and docetaxel-resistant cells was deterred most profoundly when docetaxel and gemcitabine were administered together. This study suggests that when a drug develops therapeutic resistance, sensitivity tests could be another option, ultimately providing insight into a novel alternative clinical strategy.

FOXM1 contributes to docetaxel resistance in castration-resistant prostate cancer by inducing AMPK/mTOR-mediated autophagy.

Docetaxel-mediated chemotherapy is the first line therapy for metastatic castration-resistant prostate cancer (CRPC) patients, but its therapeutic benefit is limited by the development of resistance. Although Forkhead box protein M1 (FOXM1) has been implicated in prostate tumorigenesis and metastasis, its role in docetaxel resistance has not been studied. Here, we showed that FOXM1 expression was upregulated in the docetaxel resistant CRPC cell lines (PC3-DR and VCaP-DR) and knockdown of FOXM1 sensitized the cells to docetaxel both in vitro and in vivo. In addition, autophagy was found to be significantly enhanced in resistant cells. Moreover, FOXM1 overexpression cells showed increased autophagic flux and higher numbers of autophagosomes. Knockdown of ATG7, beclin-1 or cotreatment with chloroquine, partly restored sensitivity to docetaxel in the FOXM1-overexpressing cells. Mechanistically, FOXM1 targeted AMPK/mTOR to activate the autophagy pathway and altered docetaxel response in CRPC. These findings identify the role of FOXM1 as well as the mechanism underlying FOXM1 action in docetaxel sensitivity and may, therefore, aid in design of CRPC therapies.

Risk Factors for Poor Survival in Metastatic Castration-resistant Prostate Cancer Treated With Cabazitaxel in Japan

Cabazitaxel (CBZ) is approved for docetaxel-resistant castration-resistant prostate cancer (CRPC). This retrospective study aimed at assessing the efficacy and prognostic markers of cabazitaxel treatment in Japanese CRPC patients. The medical records of 44 consecutive Japanese patients with CRPC who started cabazitaxel at our Institution between January 2011 and February 2019 were reviewed and statistically analysed. The median follow-up period after cabazitaxel initiation was 13.2 [interquartile range (IQR)=6.9-21.5] months. The objective response rate, median progression-free survival period, and median overall survival period (OS) were 45.5%, 4.3 months, and 20.7 months, respectively. On multivariate analysis, higher prostate-specific antigen (PSA; >100 ng/ml), lower haemoglobin (<10 g/dl), and lower number of prior docetaxel therapy cycles (<10) were predictors for shorter OS. Patients with anemia, high PSA, and lower number of docetaxel therapy cycles might have shorter survival period from introduction of cabazitaxel therapy. In addition, PSA decline might still be a useful indicator as a predictor of prognosis of the metastatic CRPC patients treated with cabazitaxel.

The efficacy and toxicity of cabazitaxel for treatment of docetaxel-resistant prostate cancer correlating with the initial doses in Japanese patients

BackgroundWe analyzed the efficacy and toxicity of cabazitaxel (CBZ) at high and low initial doses in Japanese patients with docetaxel-resistant castration-resistant prostate cancer (CRPC).MethodsWe retrospectively evaluated 118 patients who received CBZ for docetaxel-resistant CRPC in 10 university hospitals in Japan between 2014 and 2016. The rate of decrease of prostate-specific antigen (PSA), adverse events, progression-free survival (PFS), and overall survival (OS) were compared between patients receiving initially high (≥22.5 mg/m2, n = 36) and low (≤20 mg/m2, n = 80) CBZ doses. Factors associated with survival and grade 4 neutropenia were evaluated.ResultsPSA values decreased by > 50% in 22 patients (19%), with a higher frequency in the high-dose group than in the low-dose group (29 and 14%, P = 0.073). The median PFS time for the all-patient, high- and low-dose groups was 2.8 months (95% confidence interval [CI] 1.9–4.4), 2.1 months (1.2–5.5), and 3.0 months (2.0–4.4), respectively (P = 0.904). The median OS times were 16.3 months (95% CI 9.7–30.9), 30.9 months (11.8–47.4), and 10.2 months (8.6–20), respectively (P = 0.020). In multivariate analyses, PFS was significantly associated with existing bone metastasis at diagnosis (P = 0.005) and OS with PSA > 100 ng/ml (P = 0.007), hemoglobin < 12 g/dl (P = 0.030), and low initial CBZ dose (P = 0.030). Grade 4 neutropenia occurred in 53 patients (45%) and was associated with a low CBZ dose (hazard ratio 0.21, 95% CI 0.08–0.59, P = 0.002).ConclusionsCBZ at a higher initial dose may have similar response rate and response duration, but longer survival duration after treatment with higher toxicity than a lower initial dose for docetaxel-resistant CRPC in Japanese patients.

Novel CIL-102 derivatives as potential therapeutic agents for docetaxel-resistant prostate cancer

The standard-of-care treatment for metastatic prostate cancer (PCa) is androgen deprivation therapy (ADT). Nevertheless, most tumors eventually relapse and develop into lethal castration-resistant prostate cancer (CRPC). Docetaxel is a FDA-approved agent for the treatment of CRPC; however, the tumor often quickly develops resistance to this drug. Thus, there is an immediate need for novel therapies to treat docetaxel-resistant PCa. In this study, we modified the structure of CIL-102 and investigated the efficacy of the derivatives against CRPC and docetaxel-resistant PCa. These novel CIL-102 derivatives inhibit CRPC tumorigenicity, including proliferation, migration and colony formation, and importantly, selectively inhibit CRPC cell proliferation over non-cancerous prostate epithelia. Computational modeling indicated the derivatives bind to β-tubulin and immunocytochemistry revealed the depolymerization of microtubules upon treatment. Western blot analyses reveal that pro-apoptotic and anti-oxidant pathways are activated, and MitoSOX and DCF-DA analyses confirmed increased reactive oxygen species (ROS) production upon treatments. Furthermore, CIL-102 derivatives effectively reduce the proliferation of docetaxel-resistant CR PCa cell lines. Our data indicate the potential of these compounds as promising therapeutic agents for CRPC as well as docetaxel-resistant CRPC.

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.

A phase 1/2a trial of docetaxel plus ribavirin for reprogramming efficacy in patients with progressive metastatic castration resistant prostate cancer who have previously received docetaxel alone: DRREEM trial.

329 Background: We previously reported a novel cell reprogramming approach, termed drug efficacy reprogramming, as a new model for identifying candidate antitumor drugs targeting the cancer stemness-related gene network, and identified ribavirin as a candidate drug for overcoming docetaxel-resistant castration-resistant prostate cancer (CRPC). We conducted this phase 1/2a trial of docetaxel plus ribavirin for reprogramming efficacy in patients with progressive metastatic castration resistant prostate cancer who have previously received docetaxel alone. (DRREEM trial) Methods: In this clinical study, patients received intravenous docetaxel at 60-75 mg/m2is intravenously administered on Cycle1-Day1 in combination with the investigational drug (ribavirin). Docetaxel is administered at 3-week intervals (1 cycle) (total: 3 cycles). During administration, the dose may be reduced based on the subject’s condition if necessary. The primary endpoint was safety. Accessory evaluation items included prostate-specific antigen (PSA) response, objective response rate, health-related quality of lifel. Exploratory items included changes in CTC count, cfDNA, and exosome. Patients with progressive CRPC based on PSA and/or radiographic criteria, performance status (PS) 0–1, and normal renal and hepatic function were eligible. Results: Six patients were enrolled in this study; average age was 71.7±4.2. Average serum PSA concentration was 100.1±128.0 ng/ml (range: 3.0-336.8). The median cycle of docetaxel received before the study was 6 cycles. Safety: Grade 3/4 adverse events requiring dose modification were not observed. Two patients showed PSA reduction. Three patients showed stable disease. Changes in the blood concentrations of ribavirin, docetaxel, and prednisolone were within normal range. Conclusions: This combination of ribavirin with docetaxel was well tolerated with a promising response rate that justifies further investigations in docetaxel-resistant CRPC. This clinical study provides a useful drug re-positioning model in the area of translational medicine. Clinical trial information: UMIN000021107.