Chemoresistant Prostate Cancer Research Articles

Regulation of eIF4F Translation Initiation Complex by the Peptidyl Prolyl Isomerase FKBP7 in Taxane-resistant Prostate Cancer.

Targeted therapies that use the signaling pathways involved in prostate cancer are required to overcome chemoresistance and improve treatment outcomes for men. Molecular chaperones play a key role in the regulation of protein homeostasis and are potential targets for overcoming chemoresistance.Experimental Design: We established 4 chemoresistant prostate cancer cell lines and used image-based high-content siRNA functional screening, based on gene-expression signature, to explore mechanisms of chemoresistance and identify new potential targets with potential roles in taxane resistance. The functional role of a new target was assessed by in vitro and in vivo silencing, and mass spectrometry analysis was used to identify its downstream effectors. We identified FKBP7, a prolyl-peptidyl isomerase overexpressed in docetaxel-resistant and in cabazitaxel-resistant prostate cancer cells. This is the first study to characterize the function of human FKBP7 and explore its role in cancer. We discovered that FKBP7 was upregulated in human prostate cancers and its expression correlated with the recurrence observed in patients receiving docetaxel. FKBP7 silencing showed that FKBP7 is required to maintain the growth of chemoresistant cell lines and chemoresistant tumors in mice. Mass spectrometry analysis revealed that FKBP7 interacts with eIF4G, a component of the eIF4F translation initiation complex, to mediate the survival of chemoresistant cells. Using small-molecule inhibitors of eIF4A, the RNA helicase component of eIF4F, we were able to kill docetaxel- and cabazitaxel-resistant cells. Targeting FKBP7 or the eIF4G-containing eIF4F translation initiation complex could be novel therapeutic strategies to eradicate taxane-resistant prostate cancer cells.

Cotargeting Plk1 and androgen receptor enhances the therapeutic sensitivity of paclitaxel-resistant prostate cancer.

Backgrounds:Despite the clinical success of taxanes, they still have limitations, such as chemoresistance. To overcome the limitations of paclitaxel, genetic alterations and targeting effects of altered genes were observed in paclitaxel-resistant cancer. Because paclitaxel-resistant cancer shows high levels of Plk1, a promising target in chemotherapy, the effectiveness of Plk1 inhibitors in paclitaxel-resistant cancer cells has been investigated.Methods:Paclitaxel-resistant cancer cells were developed by exposure of stepwise escalating levels of paclitaxel. Genetic alterations were detected by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and immunoblotting. Using a cell viability assay, combined targeting effects for Plk1 and androgen receptor (AR) were determined. Clinical data were analyzed to understand the relationship between Plk1 and AR in prostate cancer patients.Results:Treatment with Plk1 inhibitors markedly reduced the expression of MDR1, MRP1, and Plk1 in the paclitaxel-resistant cancer. Among Plk1 inhibitors, genistein, recently found as a direct Plk1 inhibitor, tended to be more effective in the paclitaxel-resistant prostate cancer than the parental cancer cells, which was related to the suppression of the AR, as well as inhibition of Plk1 activity. A combination of Plk1 inhibitors and AR antagonist bicalutamide exhibited a synergistic effect in LNCaPTXR, as well as LNCaP cells, by inhibiting Plk1 and AR. Analysis of clinical data provides evidence for the relevance between Plk1 and AR in prostate cancer patients, showing that Plk1 and AR are strong predictors of poor survival rates.Conclusions:We suggest that cotargeting Plk1 and AR would be effective in advanced chemoresistant prostate cancer cells to overcome the limitations associated with paclitaxel.

Abstract 5902: RNA sequencing analysis of taxane-resistant prostate cancer cells reveals potential candidate genes for therapeutic targeting

Abstract There is no cure for metastatic prostate cancer (PCa), the most commonly diagnosed cancer and third leading cause of cancer mortality among U.S. men. Patients with advanced PCa often develop resistance to conventional therapies resulting in metastatic castration-resistant PCa (mCRPC). Docetaxel (DTX) chemotherapy is the standard-of-care for mCRPC, followed by secondary chemotherapy with cabazitaxel (CBZ), yet chemoresistance and death inevitably occurs. While pharmacological agents targeting single survival pathways have shown promise at the bench side, clinical trials have failed. This is mostly due to the presence of redundant pathways utilized by PCa tumor cells to maintain chemoresistance. Therefore, we are interested in further defining molecular pathways associated with DTX-resistance in order to identify novel therapeutic targets to overcome this resistance. We hypothesized that RNA sequencing (RNAseq) analysis comparing the transcriptomes of chemosensitive and chemoresistant mCRPC cells will reveal potential targets for combinatorial therapies. RNAseq analysis of DTX-sensitive and -resistant PC3 and DU145 cells revealed a number of differentially regulated genes that were either unique to each cell line or common to both. We found that dipeptidyl peptidase 4 (DPP4), fatty acid binding protein 5 (FABP5), nestin (NES), and tetraspanin 8 (TSPAN8) were among the top most robustly and significantly upregulated genes in both DTX-resistant PC3 and DU145 cell lines. In-house qPCR studies validated the RNAseq results for these genes, and Western blotting analysis showed increased expression of these proteins in DTX-resistant cells, which correlated with increased mRNA expression. Interestingly, NES, TSPAN8, DPP4 are genes associated with development of the cancer stem-cell (CSC) phenotype. Other genes associated with CSCs were also upregulated in the DTX-resistant cells, suggesting that in vitro selection of mCRPC cells for DTX resistance may enrich for CSCs. This novel finding is consistent with recent studies implicating CSCs in PCa chemoresistance. Further analysis and validation of our RNAseq data will increase our understanding of DTX-resistance mechanisms in PCa cells, including the yet undefined functional role of CSC-related genes. These studies also reveal potential therapeutic targets to circumvent DTX resistance in PCa. Citation Format: Christina K. Cajigas-Du Ross, Leanne Woods-Burnham, Joshua Ramirez, Xin Chen, Charles Wang, Carlos A. Casiano. RNA sequencing analysis of taxane-resistant prostate cancer cells reveals potential candidate genes for therapeutic targeting [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5902.

Abstract 5896: Downregulation of calcium-sensing receptor enhances MET-mediated prostate cancer chemosensitivity

Abstract Chemoresistance, in various types of cancer including prostate cancer (PCa), is one of the major challenges in cancer therapy. Others and our lab have reported that the epithelial-to-mesenchymal transition (EMT) plays critical roles in development of chemoresistance and metastasis. The calcium-sensing receptor (CaSR) plays important roles in maintenance of calcium homeostasis and altered CaSR expression, and functional changes are involved in certain diseases, including tumorigenesis and its development. However, the mechanism of CaSR in PCa chemoresistance remains unclear. In current study, the CaSR expression level was detected in parental PC3 and DU145 cells and their chemoresistant PC3-TxR and DU145-TxR cells. We found the CaSR was highly expressed in chemoresistant PC3-TxR and DU145-TxR cells. Then by using siRNA technique, CaSR expression was transiently knocked down in chemoresistant cells. The tumor cell migration, invasion, and colony formation were evaluated by wound healing, transwell, and colony formation assays, respectively. The drug sensitivity was evaluated using MTS assay. We observed that the abilities of tumor cell migration and invasion were significantly diminished in CaSR knockdown PC3-TxR and DU145-TxR cells. The ability of colony formation was also reduced. Interestingly, the sensitivity to paclitaxel was reactivated in CaSR knockdown PC3-TxR and DU145-TxR cells. We finally observed that E-cadherin expression was increased in CaSR knockdown chemoresistant cells, and Snail, Vimentin and β-catenin were decreased; these results are consistent with EMT phenotypic alterations. Taken together, we conclude that downregulation of CaSR enhances MET-mediated prostate cancer chemosensitivity. Supported by NSFC Key Project 81130046; NSFC projects 81773146,81272415; JCYJ20170412152943794, JCYJ20170412154619484, JCYJ20170307105128101, JCYJ2017030711041760; Guangxi Key Projects 2013GXNSFEA053004. Citation Format: Luying Huang, Fuhao Wang, Haibo Tong, Atsushi Mizokami, Evan T. Keller, Yi Lu, Jian Zhang. Downregulation of calcium-sensing receptor enhances MET-mediated prostate cancer chemosensitivity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5896.

Drugging drug resistance with bystander-assisted immunotherapy.

Abstract Acquired drug resistance is a longstanding challenge that reduces the efficacy of chemotherapeutic drugs. In contrast, the efficacies of modern immunotherapies do not directly correlate to the prevalence of chemo-resistant phenotypes. Here we improve upon this paradigm by targeting the action of immunotherapeutics to two general mechanisms of acquired drug resistance: irregular metabolism and drug efflux mediated by the ABC superfamily of transport proteins. In chemo-resistant prostate cancer cells, we demonstrate that these two mechanisms act in concert to selectively metabolize our newly developed class of enzyme-directed prodrug to the Toll-Like Receptor 7 immunotherapeutic Imiquimod. Following metabolism, liberated Imiquimod undergoes drug efflux to the extracellular space where it activates bystander immune cells in local proximity. In-vitro, we characterize this process of Bystander-Assisted ImmunoTherapy (BAIT) using an AT3B-1 chemo-resistant prostate cancer model with RAW-Blue and JAWSII reporter immune cell lines. Co-culture of AT3B-1 cancer cells with reporter immune cells results in immunogenicity mediated selectively by cancer cells. This results in enhanced NF-κB transcription, expression of cell surface markers, and secreted cytokines indicative of a cell-mediated immune response. Our prodrug is non-immunogenic with healthy cells alone and the enzyme-directing groups are stable for over 3 days in serum. Taken together, our results demonstrate that BAIT co-opts common mechanisms of drug resistance to elicit immunogenicity mediated by cancer cells themselves. We anticipate that BAIT will find use as a new mechanism of action that exploits drug resistant phenotypes to generate an immune response.

Selective eradication of cancer displaying hyperactive Akt by exploiting the metabolic consequences of Akt activation.

Akt activation in human cancers exerts chemoresistance, but pan-Akt inhibition elicits adverse consequences. We exploited the consequences of Akt-mediated mitochondrial and glucose metabolism to selectively eradicate and evade chemoresistance of prostate cancer displaying hyperactive Akt. PTEN-deficient prostate cancer cells that display hyperactivated Akt have high intracellular reactive oxygen species (ROS) levels, in part, because of Akt-dependent increase of oxidative phosphorylation. High intracellular ROS levels selectively sensitize cells displaying hyperactive Akt to ROS-induced cell death enabling a therapeutic strategy combining a ROS inducer and rapamycin in PTEN-deficient prostate tumors in mouse models. This strategy elicited tumor regression, and markedly increased survival even after the treatment was stopped. By contrast, exposure to antioxidant increased prostate tumor progression. To increase glucose metabolism, Akt activation phosphorylated HK2 and induced its expression. Indeed, HK2 deficiency in mouse models of Pten-deficient prostate cancer elicited a marked inhibition of tumor development and extended lifespan.

HSP60 IS A NOVEL TARGET IN LETHAL PROSTATE CANCER

Currently, various therapeutic agents exist in the clinic to treat aggressive prostate cancer (PCa) by modulating androgen receptor (AR) activity, which is one of the main drivers of prostate cancer. However, resistance to these agents nearly always occurs and results in the production of a more aggressive form of cancer, typically referred to as castrate‐recurrent prostate cancer (CRPC). Patients within a subset of CRPC have tumors that undergo neuroendocrine (NE) transdifferentiation (NE‐CRPC). NE‐CRPC is poorly differentiated, readily metastasizes and is resistant to androgen deprivation therapy (ADT). Therefore, it is essential that alternative therapeutic targets that do not solely rely on AR modulation be better characterized for patients with this advanced disease.The mitochondrial unfolded protein response (mitoUPR) is responsible for maintaining mitochondrial quality control by preserving the integrity of the mitochondria in response to numerous stressors including hypoxia and chemotherapeutic agents. MitoUPR activation is a hallmark sign of mitochondrial dysfunction. Heat Shock Protein60 (Hsp60) is a mitochondrial chaperone and is a key player of the mitoUPR. Thus, we hypothesize that inhibition of Hsp60 renders aggressive NE‐like cells susceptible to stress, thereby alleviating tumorigenicity. We observed that protein expression of Hsp60 was upregulated in tumors derived from NE models such as the transgenic adenocarcinoma of mouse prostate tumor (TRAMP), DKO (pten−/−/rb1−/−) and TKO (rb1−/−/pten−/−/p53−/−) when compared to wild‐type controls. Transcript levels of HSPD1 positively correlated to Gleason Score and are upregulated in patients with NE‐like phenotypes from TCGA. Additionally, Hsp60 expression associated with severe mitochondrial dysfunction in TRAMP tumors. Specifically, mito‐nuclear imbalances and defects in oxidative phosphorylation activity were observed. Lastly, Inhibition of Hsp60 significantly increased sensitivity of NE cells to mitotoxic stressors such as hypoxia and the chemotherapeutic agent cisplatin.Together, our data indicate that inactivation of Hsp60 sensitizes NE cells to mitotoxic stress and has clinical and therapeutic relevance. Specifically, the use of cisplatin in small cell CRPC has been shown to have minimal effect, yet we determined that inhibition of Hsp60 sensitizes PCa cells to cisplatin. We envision that inactivation of Hsp60 will alleviate tumor burden by itself, but will also enhance sensitivity of chemo‐resistant PCa cells. Additionally, inhibition of Hsp60 may enhance therapeutic potential of anticancer agents in other types of cancers that overexpress Hsp60 including liver, lung and colon cancers. Thus, Hsp60 is a novel target in prostate cancers with NE‐like differentiation.Support or Funding InformationThis work was supported in part by the National Cancer Institute of the National Institutes of Health under Award no. R01CA160685This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Hyper-acetylation contributes to the sensitivity of chemo-resistant prostate cancer cells to histone deacetylase inhibitor Trichostatin A.

Therapeutic agents are urgently needed for treating metastatic castration‐refractory prostate cancer (mCRPC) that is unresponsive to androgen deprivation and chemotherapy. Our screening assays demonstrated that chemotherapy‐resistant prostate cancer (PCa) cells are more sensitive to HDAC inhibitors than paired sensitive PCa cells, as demonstrated by cell proliferation and apoptosis in vitro and in vivo. Kinetic study revealed that TSA‐induced apoptosis was significantly dependent on enhanced transcription and protein synthesis in an early stage, which subsequently caused ER stress and apoptosis. ChIP analysis indicated that TSA increased H4K16 acetylation, promoting ER stress gene transcription. The changes in Ac‐H4K16, ATF3 and ATF4 were also validated in TSA‐treated animals. Further study revealed the higher enzyme activity of HDACs and an increase in acetylated proteins in resistant cells. The higher nucleocytoplasmic acetyl‐CoA in resistant cells was responsible for elevated acetylation status of protein and a more vigorous growth state. These results strongly support the pre‐clinical application of HDAC inhibitors for treating chemotherapy‐resistant mCRPC.

PrLZ increases prostate cancer docetaxel resistance by inhibiting LKB1/AMPK-mediated autophagy.

Rationale: Docetaxel-mediated chemotherapy is the first-line standard approach and has been determined to show a survival advantage for metastatic castration-resistant prostate cancer (mCRPC) patients. However, a substantial proportion of patients eventually becomes refractory due to drug resistance. The detailed mechanisms remain unclear. We have previously reported that Prostate Leucine Zipper (PrLZ), a specific oncogene of prostate cancer (PCa), promotes PCa cell growth at the castration-resistant stage, thus suggesting a vital role of PrLZ in the progression of CRPC. In this study, we aimed to investigate the role of PrLZ in docetaxel resistance in PCa, focusing on PrLZ-regulating autophagy pathway.Methods: Human PCa PC3, LNCaP and C4-2 cell lines were used as the model system in vitro and PCa xenografts and PrLZ-knockout mice were used as the model system in vivo. Docetaxel-induced cell death and apoptosis in PCa were determined by MTT and flow cytometry assay. The role of PrLZ on the regulation of autophagy and liver kinase B1/AMP-activated protein kinase (LKB1/AMPK) signaling pathway was analyzed using immunoblotting, immunoprecipitation, siRNA silencing and plasmid overexpression.Results: PrLZ increased docetaxel-mediated drug resistance both in vitro and in vivo. Mechanistic dissection revealed that PrLZ interacted with LKB1 and further inhibited the activation of LKB1/AMPK signals, which negatively contributed to the induction of autophagy. Moreover, PrLZ/LKB1-mediated autophagy conferred resistance to docetaxel-induced cell death and apoptosis both in vitro and in vivo.Conclusion: These findings identify a novel role of PrLZ in autophagy manipulation and provide new insight into docetaxel chemoresistance in PCa, suggesting a new strategy for treating mCRPC by targeting this newly identified signaling pathway.

Quercetin reverses the doxorubicin resistance of prostate cancer cells by downregulating the expression of c-met.

Chemotherapy is an irreplaceable treatment for prostate cancer. However, the acquisition of chemoresistance is a common and critical problem that requires urgent solutions for the effective treatment of this disease. The aim of the present study was to determine whether the combination of quercetin with doxorubicin reversed the resistance of prostate cancer cells to doxorubicin-based therapy. A prostate cancer (PC)3 cell line (PC3/R) with acquired doxorubicin-resistance was established. A significant drug-resistance to doxorubicin and high activation of the phosphoinositide 3-kinase/protein kinase-B (PI3K/AKT) pathway in PC3/R cells, compared with normal PC3 cells, was demonstrated. Notably, combination treatment of doxorubicin with quercetin significantly promoted the doxorubicin-induced apoptosis in PC3/R cells through the mitochondrial/reaction oxygen species pathway. In PC3/R cells, a significant upregulation of tyrosine-protein kinase-met (c-met) was observed compared with nromal PC3 cells. However, the response to quercetin treatment in PC3/R cells inhibited c-met expression and the downstream PI3K/AKT pathway. In addition, induced expression of c-met rescued quercetin-promoted apoptosis in PC3/R cells treated with doxorubicin. The results of the present study indicated that quercetin is able to reverse prostate cancer cell doxorubicin resistance by downregulating the expression of c-met. It may represent a potential strategy for reversing the chemoresistance of prostate cancer.

MiR-199a suppresses prostate cancer paclitaxel resistance by targeting YES1.

Prostate cancer chemoresistance is a major contributor to the poor survival of patients. MicroRNAs (miRNAs) play an important role in regulating cancer resistance. Here we aim to explore the role and mechanism of miR-199a in regulating prostate cancer resistance. MiR-199a expressions in human prostate cancer tissues and cell lines were investigated with real-time PCR (RT-PCR). MiR-199a was ectopically overexpressed in PC3 cells, and resistance to paclitaxel (PTX) was evaluated consequently. The interaction between miR-199a and the oncogene Yamaguchi sarcoma viral homolog 1 (YES1) was assessed after miR-199a overexpression. YES1 was ectopically overexpressed, followed by evaluation of PTX resistance. The efficacy of miR-199a as a therapeutic agent was also investigated in vivo. Downregulation of miR-199a was characteristic of prostate cancer, particularly recurrent cancers. MiR-199a was suppressed in PTX-resistant cell line. Overexpression of miR-199a inhibited PTX resistance. YES1 was a target of miR-199a, and overexpression of YES1 reversed the effect of miR-199a in suppressing PTX resistance. In vivo, miR-199a increased tumor PTX sensitivity. The downregulation of miR-199a contributes to PTX resistance in prostate cancer. YES1 mediates the regulation of miR-199a in prostate cancer PTX resistance. This miR-199a replacement therapy has potential to overcome PTX resistance.

RBPJ contributes to acquired docetaxel resistance in prostate cancer cells

Our previous work has shown that depletion of recombination signal-binding protein J (RBPJ) results in reduced cell growth in prostate cancer cells. In this study, we aimed to investigate the function of RBPJ in the chemoresistance of prostate cancer. The expression of RBPJ was quantified in docetaxel-resistant and parental prostate cancer cells. Loss- and gain-of-function experiments were conducted to explore the regulatory role of RBPJ in prostate cancer sensitivity to docetaxel. The pro-apoptotic effect of RBPJ silencing was checked in docetaxel-resistant prostate cancer cells. We found that docetaxel-resistant PC3-DR and DU145-DR cells expressed 3–5-fold high levels of RBPJ than parental PC3 and DU145 cells. Short hairpin RNA-mediated knockdown of RBPJ inhibited cell proliferation and colony formation and reversed docetaxel resistance in docetaxel-resistant prostate cancer cells. In contrast, overexpression of RBPJ promoted cell growth, colony formation, and docetaxel resistance in parental prostate cancer cells. Downregulation of RBPJ induced apoptosis in docetaxel-resistant cells, which was accompanied by enhanced cleavage of caspase-3. In addition, RBPJ silencing or overexpression markedly modulated the expression of the Bcl-2 family members including Bcl-2, Bcl-xL, Mcl-1, Bax, and Bak. Altogether, RBPJ contributes to acquisition of docetaxel resistance in prostate cancer cells and may thus represent a potential target for overcoming chemotherapeutic resistance in this malignancy.

Abstract 851: Reversion of epithelial to mesenchymal transition under androgen deprived conditions promotes chemoresistance in prostate cancer

Abstract Introduction: Metastatic prostate cancer (PC) is one of the leading causes of cancer related male deaths in Australia. Radical prostatectomy followed by androgen deprivation therapy (ADT) upon recurrence of disease is the conventional form of treatment. Upon relapse, administration of androgen targeted therapy (ATT) and subsequent chemotherapy improves quality of life and overall survival of patients. However, it is not curative and the disease ultimately progresses. Epithelial to mesenchymal transition (EMT) is a process that can facilitate tumour growth and progression to metastasis and has also been associated with chemoresistance. We therefore aim to determine how EMT and resultant reversal of this state (MET) influences the chemoresistance profile of PC cells and further determine if and how specific genetic regulators are involved in this process. Methods: Doxycycline hyclate (Dox) inducible Snai1 and Zeb1, LNCaP EMT models were used to determine the effect of the EMT status in response to a clinically relevant panel of drugs using cellular and molecular assays. All assays were performed in both fetal bovine serum (FBS) and charcoal stripped serum (CSS) supplemented culture media. CSS media was used to mimic an ADT-like environment. Using 2D monolayer and 3D spheroid [Happy Cell©] formats. Cells were induced for 5 days of EMT in the presence of Dox and were allowed to revert for 14 days of MET (absence of Dox) before the drug assays were carried out over a period of 72 hours. Live/dead cell staining was used to determine viability percentage in both models. Flow cytometry was used for 2D assays and 3D assays were analysed using either a GE IN Cell Analyzer live cell imaging system or confocal microscopy. Results: Induction of Snai1 and Zeb1 induced an elongated cell shape and increased mRNA and protein expression of mesenchymal markers such as vimentin during EMT. This was associated with a decrease in expression of epithelial markers E-cadherin and EpCam,. An eventual drop in the expression levels of vimentin at around 14 days of dox removal along with an increase in epithelial markers was observed. These changes were also associated with a gradual reversal in the phenotype of these cells. However, this reversal in phenotype and expression of EMT markers was not as striking in cells grown in CSS compared to cells in FBS media (both 2D and 3D). Mitoxantrone concentration-response assays in a 2D format showed that cells that undergo a subsequent MET are more chemoresistant than uninduced or EMT cells when grown in CSS media. This was however, not observed in cells undergoing similar transitions in the FBS media. Conclusions: The chemoresistance profile of prostate cancer cells is altered by their EMT status. This effect is enhanced in the context of ADT. Cells that revert back to a more epithelial phenotype after undergoing an EMT exhibit higher resistance towards chemotherapy drugs. Citation Format: Akanksha Upadhyaya, Nataly Stylianou, Anthony M. Davies, Sarah-Louise Ryan, Brett G. Hollier, Elizabeth D. Williams. Reversion of epithelial to mesenchymal transition under androgen deprived conditions promotes chemoresistance in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 851. doi:10.1158/1538-7445.AM2017-851

Abstract 4167: Co-targeting the stress survival proteins LEDGF/p75 and clusterin to resensitize chemoresistant prostate cancer cells to docetaxel

Abstract Prostate cancer (PCa) is the most commonly diagnosed male cancer and the second leading cause of cancer deaths in U.S. men. Late-stage PCa is characterized by metastasis followed by castration resistant PCa (mCRPC), for which there is no cure. Docetaxel (DTX) is the first-line chemotherapeutic drug for late-stage PCa, and resistance to this drug is inevitable. Our group has previously reported the elevated expression of the stress oncoprotein lens epithelium-derived growth factor (LEDGF/p75) in PCa cells and tissues, and this upregulation promotes resistance to DTX-induced non-apoptotic lysosomal cell death. Another stress oncoprotein, clusterin (CLU), is also upregulated in DTX-resistant PCa, and protects cells from DTX-induced mitochondrial-triggered apoptosis. In this study we observed that targeting LEDGF/75 with siRNAs partially resensitized DTX-resistant (DR) mCRPC cell lines to DTX treatment in clonogenic assays. Similarly, other groups have shown that knockdown of CLU in mCRPC-DR cells resensitizes them to treatment with taxanes. However, recent clinical trials targeting CLU have not been effective, suggesting that blocking this protein alone is insufficient to overcome DTX resistance. We hypothesized that simultaneous knockdown or inhibition of both LEDGF/p75 and CLU could be more effective in resensitizing chemoresisant PCa cells to taxane therapy than targeting each protein alone. Because of LEDGF/p75’s role as a stress transcription co-activator, we first sought to determine if this protein contributes to CLU expression by performing siRNA-mediated knockdown of LEDGF/p75 on the taxane-sensitive and -resistant mCRPC cell lines PC3 and DU145. Western blotting revealed that CLU protein expression was not downregulated by LEDGF/p75 knockdown, compared to treatment with scrambled siRNA control. Similarly, CLU knockdown in these cell lines did not downregulate LEDGF/p75. These results indicated that CLU expression is independent of LEDGF/p75, consistent with previous observations that these two proteins promote chemoresistance via distinct, possibly redundant, mechanisms. We then performed siRNA-mediated knockdown of LEDGF/p75 and CLU, separately or together, in PC3 DR and DU145 DR cells, in the presence and absence of varying concentrations of DTX. MTT and clonogenic assays were used to determine cell death viability and clonogenicity, respectively. Simultaneous knockdown of LEDGF/p75 and CLU, in combination with DTX treatment, resulted in increased sensitization to DTX than single knockdown of each protein. These findings suggest that co-targeting LEDGF/p75 and CLU could be an effective strategy for ameliorating chemoresistance in mCRPC, and set the foundation for combinatorial therapies targeting multiple stress pathways linked to taxane resistance together with taxane chemotherapy for treatment of late-stage PCa. Citation Format: Christina K. Cajigas-Du Ross, Leslimar Rios-Colon, Leanne Woods-Burnham, Carlos A. Casiano. Co-targeting the stress survival proteins LEDGF/p75 and clusterin to resensitize chemoresistant prostate cancer cells to docetaxel [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4167. doi:10.1158/1538-7445.AM2017-4167

Abstract 1462: Exosome-derived miR-429 contributes to prostate cancer chemoresistance

Abstract Prostate cancer (PCa) is the most common cancer in men and the 2nd leading cause of cancer-related death in the United States. Certain miRNAs, act as tumor promoting or inhibiting factors in tumor development, function primarily by targeting specific mRNAs for degradation or inhibition of translation of their targeted proteins. Emerging evidence indicates that exosomes play a key role in tumor-host crosstalk, and exosome secretion, composition, and functional capacity are altered as tumors progress to aggressive phenotypes. From our prior report, we have constructed a potential network of 29 deregulated miRNAs, including 19 up and 10 down, in exosome samples derived from two kinds of paclitaxel resistant PCa cells (PC3-TXR and DU145-TXR) compared with their parental cells (PC3 and DU145). In this study, to further determine the roles of certain miRNAs derived from exosomes in chemoresistant cells, we firstly confirmed the lower expression level of miR-429 miRNA in the exosomes derived from the paclitaxel resistant PCa cells compared to their parental cells. In addition, when the miR-429 was over-expressed in PC3-TXR and DU145-TXR cells by stable transfection, epithelial-to-mesenchymal transition (EMT) markers significantly altered together with morphological changes. Overexpression of miR-429 decreased the tumor cell migration and invasion that associated with inactivation of PI-3K/AKT and Notch signaling pathways since the pathway specific inhibitors significantly diminished the transfected tumor cell migration and invasion. Finally, we found that overexpression of miRNA-429 significantly enhanced chemo-sensitivity of the chemoresistant PCa cells. These data suggested that exosome-derived miR-429 contributes to PCa chemoresistance and miR-429 may become a potential therapeutic target for PCa patients. This work was supported by Natural Science Foundation of China (NSFC) Key Project (81130046); NSFC (81171993; 81272415); Guangxi Projects of China (2013GXNSFEA053004; 2012GXNSFCB053004; 2013GXNSFBA019177; 1355004-5; 201201ZD004; GZPT13-35; 14122008-22; 11-031-05-K2; KY2015YB057; 14-045-12-K2). Note: This abstract was not presented at the meeting. Citation Format: Yang Liu, Liye Xie, Jing Li, Atsushi Mizokami, Evan T. Keller, Yi Lu, Jian Zhang. Exosome-derived miR-429 contributes to prostate cancer chemoresistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1462. doi:10.1158/1538-7445.AM2017-1462

Inhibition of Autophagy Promotes Salinomycin-Induced Apoptosis via Reactive Oxygen Species-Mediated PI3K/AKT/mTOR and ERK/p38 MAPK-Dependent Signaling in Human Prostate Cancer Cells.

Recently, the interplay between autophagy and apoptosis has become an important factor in chemotherapy for cancer treatment. Inhibition of autophagy may be an effective strategy to improve the treatment of chemo-resistant cancer by consistent exposure to chemotherapeutic drugs. However, no reports have clearly elucidated the underlying mechanisms. Therefore, in this study, we assessed whether salinomycin, a promising anticancer drug, induces apoptosis and elucidated potential antitumor mechanisms in chemo-resistant prostate cancer cells. Cell viability assay, Western blot, annexin V/propidium iodide assay, acridine orange (AO) staining, caspase-3 activity assay, reactive oxygen species (ROS) production, and mitochondrial membrane potential were assayed. Our data showed that salinomycin alters the sensitivity of prostate cancer cells to autophagy. Pretreatment with 3-methyladenine (3-MA), an autophagy inhibitor, enhanced the salinomycin-induced apoptosis. Notably, salinomycin decreased phosphorylated of AKT and phosphorylated mammalian target of rapamycin (mTOR) in prostate cancer cells. Pretreatment with LY294002, an autophagy and PI3K inhibitor, enhanced the salinomycin-induced apoptosis by decreasing the AKT and mTOR activities and suppressing autophagy. However, pretreatment with PD98059 and SB203580, an extracellular signal-regulated kinases (ERK), and p38 inhibitors, suppressed the salinomycin-induced autophagy by reversing the upregulation of ERK and p38. In addition, pretreatment with N-acetyl-l-cysteine (NAC), an antioxidant, inhibited salinomycin-induced autophagy by suppressing ROS production. Our results suggested that salinomycin induces apoptosis, which was related to ROS-mediated autophagy through regulation of the PI3K/AKT/mTOR and ERK/p38 MAPK signaling pathways.

LncRNA HOXD-AS1 Regulates Proliferation and Chemo-Resistance of Castration-Resistant Prostate Cancer via Recruiting WDR5

Castration-resistant prostate cancer (CRPC) that occurs after the failure of androgen deprivation therapy is the leading cause of deaths in prostate cancer patients. Thus, there is an obvious and urgent need to fully understand the mechanism of CRPC and discover novel therapeutic targets. Long noncoding RNAs (lncRNAs) are crucial regulators in many human cancers, yet their potential roles and molecular mechanisms in CRPC are poorly understood. In this study, we discovered that an lncRNA HOXD-AS1 is highly expressed in CRPC cells and correlated closely with Gleason score, T stage, lymph nodes metastasis, and progression-free survival. Knockdown of HOXD-AS1 inhibited the proliferation and chemo-resistance of CRPC cells invitro and invivo. Furthermore, we identified several cell cycle, chemo-resistance, and castration-resistance-related genes, including PLK1, AURKA, CDC25C, FOXM1, and UBE2C, that were activated transcriptionally by HOXD-AS1. Further investigation revealed that HOXD-AS1 recruited WDR5 to directly regulate the expression of target genes by mediating histone H3 lysine 4 tri-methylation (H3K4me3). In conclusion, our findings indicate that HOXD-AS1 promotes proliferation, castration resistance, and chemo-resistance in prostate cancer by recruiting WDR5. This sheds a new insight into the regulation of CRPC by lncRNA and provides a potential approach for the treatment of CRPC.

Bone Microenvironment Changes in Latexin Expression Promote Chemoresistance.

Although docetaxel is the standard of care for advanced prostate cancer, most patients develop resistance to docetaxel. Therefore, elucidating the mechanism that underlies resistance to docetaxel is critical to enhance therapeutic intervention. Mining cDNA microarray from the PC-3 prostate cancer cell line and its docetaxel-resistant derivative (PC3-TxR) revealed decreased latexin (LXN) expression in the resistant cells. LXN expression was inversely correlated with taxane resistance in a panel of prostate cancer cell lines. LXN knockdown conferred docetaxel resistance to prostate cancer cells in vitro and in vivo, whereas LXN overexpression reduced docetaxel resistance in several prostate cancer cell lines. A mouse model of prostate cancer demonstrated that prostate cancer cells developed resistance to docetaxel in the bone microenvironment, but not the soft tissue microenvironment. This was associated with decreased LXN expression in prostate cancer cells in the bone microenvironment compared with the soft tissue microenvironment. It was identified that bone stromal cells decreased LXN expression through methylation and induced chemoresistance in prostate cancer in vitro These findings reveal that a subset of prostate cancer develops docetaxel resistance through loss of LXN expression associated with methylation and that the bone microenvironment promotes this drug resistance phenotype.Implications: This study suggests that the LXN pathway should be further explored as a viable target for preventing or reversing taxane resistance in prostate cancer. Mol Cancer Res; 15(4); 457-66. ©2017 AACR.

Down-regulation of E-cadherin enhances prostate cancer chemoresistance via Notch signaling

BackgroundThe chemoresistance of prostate cancer (PCa) is invariably associated with the aggressiveness and metastasis of this disease. New emerging evidence indicates that the epithelial-to-mesenchymal transition (EMT) may play pivotal roles in the development of chemoresistance and metastasis. As a hallmark of EMT, E-cadherin is suggested to be a key marker in the development of chemoresistance. However, the molecular mechanisms underlying PCa chemoresistance remain unclear. The current study aimed to explore the association between EMT and chemoresistance in PCa as well as whether changing the expression of E-cadherin would affect PCa chemoresistance.MethodsParental PC3 and DU145 cells and their chemoresistant PC3-TxR and DU145-TxR cells were analyzed. PC3-TxR and DU145-TxR cells were transfected with E-cadherin-expressing lentivirus to overexpress E-cadherin; PC3 and DU145 cells were transfected with small interfering RNA to silence E-cadherin. Changes of EMT phenotype-related markers and signaling pathways were assessed by Western blotting and quantitative real-time polymerase chain reaction. Tumor cell migration, invasion, and colony formation were then evaluated by wound healing, transwell, and colony formation assays, respectively. The drug sensitivity was evaluated using MTS assay.ResultsChemoresistant PC3-TxR and DU145-TxR cells exhibited an invasive and metastatic phenotype that associated with EMT, including the down-regulation of E-cadherin and up-regulation of Vimentin, Snail, and N-cadherin, comparing with that of parental PC3 and DU145 cells. When E-cadherin was overexpressed in PC3-TxR and DU145-TxR cells, the expression of Vimentin and Claudin-1 was down-regulated, and tumor cell migration and invasion were inhibited. In particular, the sensitivity to paclitaxel was reactivated in E-cadherin-overexpressing PC3-TxR and DU145-TxR cells. When E-cadherin expression was silenced in parental PC3 and DU145 cells, the expression of Vimentin and Snail was up-regulated, and, particularly, the sensitivity to paclitaxel was decreased. Interestingly, Notch-1 expression was up-regulated in PC3-TxR and DU145-TxR cells, whereas the E-cadherin expression was down-regulated in these cells comparing with their parental cells. The use of γ-secretase inhibitor, a Notch signaling pathway inhibitor, significantly increased the sensitivity of chemoresistant cells to paclitaxel.ConclusionThe down-regulation of E-cadherin enhances PCa chemoresistance via Notch signaling, and inhibiting the Notch signaling pathway may reverse PCa chemoresistance.

Targeting the stress oncoprotein LEDGF/p75 to sensitize chemoresistant prostate cancer cells to taxanes.

Prostate cancer (PCa) is associated with chronic prostate inflammation resulting in activation of stress and pro-survival pathways that contribute to disease progression and chemoresistance. The stress oncoprotein lens epithelium-derived growth factor p75 (LEDGF/p75), also known as DFS70 autoantigen, promotes cellular survival against environmental stressors, including oxidative stress, radiation, and cytotoxic drugs. Furthermore, LEDGF/p75 overexpression in PCa and other cancers has been associated with features of tumor aggressiveness, including resistance to cell death and chemotherapy. We report here that the endogenous levels of LEDGF/p75 are upregulated in metastatic castration resistant prostate cancer (mCRPC) cells selected for resistance to the taxane drug docetaxel (DTX). These cells also showed resistance to the taxanes cabazitaxel (CBZ) and paclitaxel (PTX), but not to the classical inducer of apoptosis TRAIL. Silencing LEDGF/p75 effectively sensitized taxane-resistant PC3 and DU145 cells to DTX and CBZ, as evidenced by a significant decrease in their clonogenic potential. While TRAIL induced apoptotic blebbing, caspase-3 processing, and apoptotic LEDGF/p75 cleavage, which leads to its inactivation, in both taxane-resistant and -sensitive PC3 and DU145 cells, treatment with DTX and CBZ failed to robustly induce these signature apoptotic events. These observations suggested that taxanes induce both caspase-dependent and -independent cell death in mCRPC cells, and that maintaining the structural integrity of LEDGF/p75 is critical for its role in promoting taxane-resistance. Our results further establish LEDGF/p75 as a stress oncoprotein that plays an important role in taxane-resistance in mCRPC cells, possibly by antagonizing drug-induced caspase-independent cell death.