Docetaxel-resistant Cells Research Articles

FTH1P8 induces and transmits docetaxel resistance by inhibiting ferroptosis in prostate cancer

Overcoming docetaxel resistance remains a significant challenge in the management of prostate cancer. Previous studies have confirmed a link between ferroptosis and the development of docetaxel resistance. This study revealed that docetaxel-resistant prostate cancer cells presented increased FTH1P8 expression compared with docetaxel-sensitive cells. Decreasing the level of FTH1P8 counteracted docetaxel resistance and facilitated docetaxel-induced ferroptosis, which is characterized by an increase in intracellular Fe2+ concentration, lipid peroxidation levels (lipid ROS), reactive oxygen species (ROS) accumulation, malondialdehyde (MDA) production and mitochondrial damage, a decrease in the Fe3+ concentration and glutathione (GSH) content, and the ability to inhibit hydroxyl radical (·OH) and the mitochondrial membrane potential (MMP). Conversely, increasing the level of FTH1P8 had the opposite effect. A positive correlation was revealed between the expression of FTH1P8 and its parental gene FTH1 in prostate cancer tissues in The Cancer Genome Atlas (TCGA) database. Molecular investigations revealed that FTH1P8 expression increased through miR-1252–5p. Furthermore, rescue experiments confirmed that FTH1 mediated the inhibitory effect of FTH1P8 on ferroptosis. Moreover, FTH1P8 was discovered to play a role in the spread of docetaxel resistance via exosomes. Docetaxel-siRNA targeting FTH1P8 (siFTH1P8)-nanoliposomes (DOC-siFTH1P8-LIP), which can codeliver docetaxel and siFTH1P8, significantly inhibited docetaxel resistance in cells. These results indicated that FTH1P8 can function as both an indicator and a treatment target for docetaxel resistance. The use of DOC-siFTH1P8-LIP demonstrated promising therapeutic effects on docetaxel-resistant cells, suggesting a novel option for treating docetaxel-resistant prostate cancer.

Pre-existing cell subpopulations in primary prostate cancer tumors display surface fingerprints of docetaxel-resistant cells.

Docetaxel resistance is a significant obstacle in the treatment of prostate cancer (PCa), resulting in unfavorable patient prognoses. Intratumoral heterogeneity, often associated with epithelial-to-mesenchymal transition (EMT), has previously emerged as a phenomenon that facilitates adaptation to various stimuli, thus promoting cancer cell diversity and eventually resistance to chemotherapy, including docetaxel. Hence, understanding intratumoral heterogeneity is essential for better patient prognosis and the development of personalized treatment strategies. To address this, we employed a high-throughput single-cell flow cytometry approach to identify a specific surface fingerprint associated with docetaxel-resistance in PCa cells and complemented it with proteomic analysis of extracellular vesicles. We further validated selected antigens using docetaxel-resistant patient-derived xenografts in vivo and probed primary PCa specimens to interrogate of their surface fingerprint. Our approaches revealed a 6-molecule surface fingerprint linked to docetaxel resistance in primary PCa specimens. We observed consistent overexpression of CD95 (FAS/APO-1), and SSEA-4 surface antigens in both in vitro and in vivo docetaxel-resistant models, which was also observed in a cell subpopulation of primary PCa tumors exhibiting EMT features. Furthermore, CD95, along with the essential enzymes involved in SSEA-4 synthesis, ST3GAL1, and ST3GAL2, displayed a significant increase in patients with PCa undergoing docetaxel-based therapy, correlating with poor survival outcomes. In summary, we demonstrate that the identified 6-molecule surface fingerprint associated with docetaxel resistance pre-exists in a subpopulation of primary PCa tumors before docetaxel treatment. Thus, this fingerprint warrants further validation as a promising predictive tool for docetaxel resistance in PCa patients prior to therapy initiation.

Inhibition of the Sterol Regulatory Element Binding Protein SREBF-1 Overcomes Docetaxel Resistance in Advanced Prostate Cancer

Resistance to anti-androgens and chemotherapy (Cx) limits therapeutic options for patients with hormone-sensitive (mHSPC) and hormone-resistant prostate cancer (mCRPC). In this context, upregulation of the glucocorticoid receptor (GR) has been identified as a potential bypass mechanism in mCRPC, and a combination of docetaxel and mifepristone (Doc + RU-486), an inhibitor of GR, re-sensitized docetaxel-resistant cell models to Cx. This study was designed to elucidate the molecular mechanisms responsible for this phenomenon. RNA sequencing (RNA-seq) in docetaxel-resistant PCa cell models after Doc + RU-486 treatment with consecutive functional assays were performed. Expression of selected proteins was verified in prostatic tissue from prostate cancer (PCa) patients with progressive disease. Treatment with Doc + RU-486 significantly reduced cancer cell viability and RNA-Seq. revealed sterol regulatory element of binding transcription factor 1 (SREBF-1), a transcription factor of cholesterol and lipid biosynthesis, as a significantly downregulated target. Functional assays confirmed that SREBF-1 downregulation is partially responsible for this observation. In concordance, SREBF-1 knockdown and pharmacological SREBP inhibition, together with other key enzymes in the cholesterol pathway, showed similar results. Furthermore, SREBF-1 expression is significantly elevated in advanced PCa tissues, demonstrating its potential involvement in tumor progression and emerging therapy resistance. Therefore, specific inhibition of cholesterol and lipid biosynthesis might also target Cx-resistant cancer cells and represent a potential additive future therapeutic option to improve mCRPC therapy.

Exploiting epigenetic targets to overcome taxane resistance in prostate cancer

The development of taxane resistance remains a major challenge for castration resistant prostate cancer (CR-PCa), despite the effectiveness of taxanes in prolonging patient survival. To uncover novel targets, we performed an epigenetic drug screen on taxane (docetaxel and cabazitaxel) resistant CR-PCa cells. We identified BRPF reader proteins, along with several epigenetic groups (CBP/p300, Menin-MLL, PRMT5 and SIRT1) that act as targets effectively reversing the resistance mediated by ABCB1. Targeting BRPFs specifically resulted in the resensitization of resistant cells, while no such effect was observed on the sensitive compartment. These cells were successfully arrested at the G2/M phase of cell cycle and underwent apoptosis upon BRPF inhibition, confirming the restoration of taxane susceptibility. Pharmacological inhibition of BRPFs reduced ABCB1 activity, indicating that BRPFs may be involved in an efflux-related mechanism. Indeed, ChIP-qPCR analysis confirmed binding of BRPF1 to the ABCB1 promoter suggesting direct regulation of the ABCB1 gene at the transcriptional level. RNA-seq analysis revealed that BRPF1 knockdown affects the genes enriched in mTORC1 and UPR signaling pathways, revealing potential mechanisms underlying its functional impact, which is further supported by the enhancement of taxane response through the combined inhibition of ABCB1 and mTOR pathways, providing evidence for the involvement of multiple BRPF1-regulated pathways. Beyond clinical attributes (Gleason score, tumor stage, therapy outcome, recurrence), metastatic PCa databases further supported the significance of BRPF1 in taxane resistance, as evidenced by its upregulation in taxane-exposed PCa patients.

Dual inhibition of MEK and PI3Kβ/δ-a potential therapeutic strategy in PTEN-wild-type docetaxel-resistant metastatic prostate cancer.

Background: Docetaxel remains the standard treatment for metastatic castration-resistant prostate cancer (mCRPC). However, resistance frequently emerges as a result of hyperactivation of the PI3K/AKT and the MEK/ERK pathways. Therefore, the inhibition of these pathways presents a potential therapeutic approach. In this study, we evaluated the efficacy of simultaneous inhibition of the PI3K/AKT and MEK/ERK pathways in docetaxel-resistant mCRPC, both in vitro and in vivo. Methods: Docetaxel-sensitive and docetaxel-resistant mCRPC cells were treated with selumetinib (MEK1/2 inhibitor), AZD8186 (PI3Kβ/δ inhibitor) and capivasertib (pan-AKT inhibitor) alone and in combination. Efficacy and toxicity of selumetinib+AZD8186 were tested in docetaxel-resistant xenograft mice. CRISPR-Cas9 generated a PTEN-knockdown docetaxel-resistant cell model. Changes in phosphorylation of AKT, ERK and downstream targets were analyzed by Western blot. Antiapoptotic adaptations after treatments were detected by dynamic BH3 profiling. Results: PI3K/AKT and MEK/ERK pathways were hyperactivated in PTEN-wild-type (wt) docetaxel-resistant cells. Selumetinib+AZD8186 decreased cell proliferation and increased apoptosis in PTEN-wt docetaxel-resistant cells. This observation was further confirmed in vivo, where docetaxel-resistant xenograft mice treated with selumetinib+AZD8186 exhibited reduced tumor growth without additional toxicity. Conclusion: Our findings on the activity of selumetinib+AZD8186 in PTEN-wt cells and in docetaxel-resistant xenograft mice provide an excellent rationale for a novel therapeutic strategy for PTEN-wt mCRPC patients resistant to docetaxel, in whom, unlike PTEN-loss patients, a clinical benefit of treatment with single-agent PI3K and AKT inhibitors has not been demonstrated. A phase I-II trial of this promising combination is warranted.

NOTCH3 promotes docetaxel resistance of prostate cancer cells through regulating TUBB3 and MAPK signaling pathway.

Docetaxel is the preferred chemotherapeutic agent in patients with castrate-resistant prostate cancer (CRPC). However, patients eventually develop docetaxel resistance and in the absence of effective treatment options. Consequently, it is essential to investigate the mechanisms generating docetaxel resistance and develop novel alternative therapeutic targets. RNA sequencing was undertaken on docetaxel-sensitive and docetaxel-resistant prostate cancer (PCa) cells. Subsequently, chemoresistance, cancer stemness, and lipid metabolism were investigated. To obtain insight into the precise activities and action mechanisms of NOTCH3 in docetaxel-resistant PCa, immunoprecipitation, mass spectrometry, ChIP, luciferase reporter assay, cell metabolism, and animal experiments were performed. Through RNA sequencing analysis, we found that NOTCH3 expression was markedly higher in docetaxel-resistant cells relative to parental cells, and that this trend was continued in docetaxel-resistant PCa tissues. Experiments invitro and invivo revealed that NOTCH3 enhanced stemness, lipid metabolism, and docetaxel resistance in PCa. Mechanistically, NOTCH3 is bound to TUBB3 and activates the MAPK signaling pathway. Moreover, NOTCH3 was directly regulated by MEF2A in docetaxel-resistant cells. Notably, targeting NOTCH3 and the MEF2A/TUBB3 signaling axis was related to docetaxel chemoresistance in PCa. Overall, these results demonstrated that NOTCH3 fostered stemness, lipid metabolism, and docetaxel resistance in PCa via the TUBB3 and MAPK signaling pathways. Therefore, NOTCH3 may be employed as a prognostic biomarker in PCa patients. NOTCH3 could be a therapeutic target for PCa patients, particularly those who have developed docetaxel resistance.

THU502 CYR61, A Member Of The CCN Protein Family, Regulates IGF1 In Metastatic Prostate Cancer

Abstract Disclosure: G.L. Ortiz Hernandez: None. M. Walker: None. L. Woods-Burnham: None. R.A. Kittles: None. C.A. Casiano: None. S.L. Neuhausen: None. The cysteine-rich angiogenic inducer 61 (CYR61) is a member of the cellular communication network (CCN) protein family that can be induced by growth factors. CYR61-specific functions are dependent on cellular context. In prostate cancer (PCa), CYR61 contributes to cell growth and survival through its interactions with extracellular ligands, such as integrins avb3 and a6b4, in the extracellular matrix space. Interestingly, CYR61 contains an insulin-like growth factor-binding protein domain suggesting that it may interact with insulin-like growth factor-1 (IGF1). High serum levels of IGF1 are directly linked to PCa development and recently have been studied as a predictor of metastatic disease. Given the important roles that IGF1 and CYR61 play in PCa and their potential interaction, it is critical to investigate their molecular interplay. We hypothesize that the interaction of CYR61 and IGF1 contributes to aggressive tumor properties (e.g., proliferation, tumorsphere formation) in PCa. Using immunoblotting, we demonstrated that CYR61 is upregulated in the androgen receptor-negative and glucocorticoid receptor-positive (AR-/GR+) metastatic cell line, PC3, and downregulated in the docetaxel-resistant cell line PC3-DR. Furthermore, optimized knockdown of CYR61 using siRNA significantly reduced PC3 cell proliferation, viability, and prostasphere formation. To examine the underlying mechanisms associated with IGF1 signaling, we assessed the activation of the PI3/Akt and MAPK pathways in PC3 cells with CYR61 silenced. CYR61 siRNA-mediated knockdown decreased activation of the PI3/Akt pathway but did not alter the MAPK pathway. We also initiated studies to determine the co-localization of CYR61 and IGF1 using immunofluorescence microscopy (IF). Based on the initial IF observations, we will proceed to examine if their interaction is primarily intra- or extra-cellular and whether interactions differ based on AR signaling. Using both PC3 and MDA-PCa-2b, which is AR+/GR+, we will co-immunoprecipitate CYR61 with IGF1 to assess intracellular interactions and use the AVidity-based EXtracellular Interaction Screen (AVEXIS) system for extracellular interactions. The correlation between PCa aggressiveness and circulating levels of CYR61 and IGF1 is poorly studied. Therefore, our goal is to establish the contribution of CYR61-IGF1 protein-protein interaction to PCa cell proliferation and tumor aggressiveness. Presentation: Thursday, June 15, 2023

Inhibition of Ribonucleotide Reductase Induces Endoplasmic Reticulum Stress and Apoptosis, Leading to the Death of Docetaxel-resistant Prostate Cancer Cells.

The development of chemotherapy resistance in prostate cancer (PCa) patients poses a significant obstacle to disease progression. Ribonucleotide reductase is a crucial enzyme for cell division and tumor growth. Triapine, an inhibitor of ribonucleotide reductase, has shown strong anti-tumor activity in various types of cancers. However, the effect of triapine on docetaxel-resistant (DR) human PCa cells has not been explored previously. This study aimed to examine the potential anti-proliferative effects of triapine in PC3-DR (docetaxel-resistant) cells. Cell viability was determined by the MTT test, and apoptosis and cell cycle progression were analyzed by image-based cytometer. mRNA and protein expression were assessed by RT-qPCR and western blot, respectively. Triapine administration significantly reduced PC3 and PC3-DR cells' survival, while the cytotoxic effect was higher in PC3-DR cells. Cell death resulting from inhibition of ribonucleotide reductase was mediated by endoplasmic reticulum stress, induction of apoptosis, and cell cycle arrest. The findings were supported by the upregulation of caspases, Bax, Bak, P21, P27, P53, TNF-α, FAS, and FASL, and downregulation of Bcl2, Bcl-XL, cyclin-dependent kinase 2 (CDK2), CDK4, cyclins, and heat shock proteins expression. According to the data, the reduction of ABC transporter proteins and NF-ĸB expression may play a role in triapine-mediated cytotoxicity in docetaxel-resistant cells. Based on our findings, triapine emerges as a promising chemotherapeutic approach for combating docetaxel- resistant prostate cancer.

Cullin7 induces docetaxel resistance by regulating the protein level of the antiapoptotic protein Survivin in lung adenocarcinoma cells.

Lung adenocarcinoma (LUAD) is the most common subtype of non-small cell lung cancer (NSCLC). Chemotherapy resistance is the main cause of chemotherapy failure. Cullin7 (Cul7) is highly expressed in LUAD and is associated with poor prognosis. Moreover, Cul7 is abnormally overexpressed in docetaxel-resistant LUAD cells. Therefore, further exploration of the role and molecular mechanism of Cul7 in LUAD docetaxel resistance is necessary. We established docetaxel-resistant cell lines (A549DTX and H358DTX cell lines) by exposing cells to gradually increasing concentrations of docetaxel. Cell (A549, A549DTX, H358, and H358DTX cell lines) sensitivity to docetaxel was determined via a 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymmethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) assay. And then quantitative polymerase chain reaction (qPCR) and Western blotting were performed to measure the expression of Cul7 and Survivin in A549, A549DTX, H358, and H358DTX cell lines. Subsequently, we knocked down Cul7 in docetaxel-resistant cells and overexpressed Cul7 in parental cells via lentiviral transduction to further validate the correlation between Cul7 and docetaxel resistance, while exploring the molecular mechanism of docetaxel resistance it caused. Immunofluorescence and immunohistochemical (IHC) staining were also used to evaluate the expression and cellular localization of Cul7. To confirm the effect of Cul7 expression on cell apoptosis, we used flow cytometry to detect the apoptosis rate of A549 and A549DTX cells with the same drug concentration. Cul7 was highly expressed in A549DTX and H358DTX cells. However, when Cul7 expression was knocked down in A549DTX and H358DTX cells, cell sensitivity to docetaxel was significantly increased. In addition, we found that Cul7 was coexpressed with Survivin. Silencing Survivin reversed the docetaxel insensitivity caused by Cul7 overexpression. High expression of Cul7 and Survivin in docetaxel-resistant LUAD cells inhibited the intrinsic apoptosis pathway and promoted cell proliferation. Therefore, the Cul7/Survivin axis may play a role in inducing LUAD docetaxel chemoresistance. Cul7 and Survivin were both highly expressed in docetaxel-resistant LUAD cells. Our results suggest that Cul7 may inhibit apoptosis and promote the proliferation of LUAD cells by increasing the Survivin protein level, which in turn contributes to docetaxel chemoresistance in LUAD.

Functional inhibition of the RNA-binding protein HuR sensitizes triple-negative breast cancer to chemotherapy.

Chemotherapy remains the standard treatment for triple-negative breast cancer (TNBC); however, chemoresistance compromises its efficacy. The RNA-binding protein Hu antigen R (HuR) could be a potential therapeutic target to enhance the chemotherapy efficacy. HuR is known to mainly stabilize its target mRNAs, and/or promote the translation of encoded proteins, which are implicated in multiple cancer hallmarks, including chemoresistance. In this study, a docetaxel-resistant cell subline (231-TR) was established from the human TNBC cell line MDA-MB-231. Both the parental and resistant cell lines exhibited similar sensitivity to the small molecule functional inhibitor of HuR, KH-3. Docetaxel and KH-3 combination therapy synergistically inhibited cell proliferation in TNBC cells and tumor growth in three animal models. KH-3 downregulated the expression levels of HuR targets (e.g., β-Catenin and BCL2) in a time- and dose-dependent manner. Moreover, KH-3 restored docetaxel's effects on activating Caspase-3 and cleaving PARP in 231-TR cells, induced apoptotic cell death, and caused S-phase cell cycle arrest. Together, our findings suggest that HuR is a critical mediator of docetaxel resistance and provide a rationale for combining HuR inhibitors and chemotherapeutic agents to enhance chemotherapy efficacy.

MiR-138–5p inhibits prostate cancer cell proliferation and chemoresistance by targeting APOBEC3B

Docetaxel is one of the most commonly used drugs in prostate cancer (PCa) chemotherapy, but its therapeutic effect in PCa is usually limited due to its drug resistance. APOBEC3B is a DNA cytosine deaminase that can alter biological processes, including chemoresistance. APOBEC3B is upregulated in various cancers. However, the biological function and underlying regulation of APOBEC3B in PCa remain unclear. In this study, we explored the role of APOBEC3B in PCa chemoresistance and the molecular mechanism of its dysregulated expression. Our results revealed that APOBEC3B was upregulated in PCa docetaxel-resistant cells, while its knockdown significantly repressed cell proliferation and docetaxel resistance of PCa cells. Bioinformatics and luciferase report analysis showed that miR-138–5p targeted APOBEC3B. In addition, miR-138–5p overexpression impeded cell proliferation and docetaxel resistance in PCa, while miR-138–5p inhibitors reversed this process. Further studies showed that upregulation of APOBEC3B expression in docetaxel-resistant cells overexpressing miR-138–5p could desensitize PCa cells to docetaxel treatment. Taken together, miR-138–5p regulates PCa cell proliferation and chemoresistance by targeting the 3′-UTR of APOBEC3B, which may provide novel insights and therapeutic targets for the treatment of PCa.

Abstract 1687: ATPase copper transporting beta (ATP7B) contributes to acquired docetaxel resistance in human prostate cancer

Abstract Docetaxel has been the standard first-line chemotherapy for lethal metastatic prostate cancer (mPCa) since 2004, but the resistance to docetaxel treatment is common. The molecular mechanisms of docetaxel resistance remain largely unknown and could be amenable to interventions that mitigate resistance. We have recently discovered that several docetaxel resistant mPCa cell lines exhibit less uptake of cellular copper and express markedly higher levels of ATP7B protein without significant changes in the protein expression levels of other copper transporters, such as ATPase copper transporting alpha (ATP7A) and high affinity copper uptake protein 1 (CTR1). Knock-down of ATP7B by silencing RNAs sensitized docetaxel resistant-mPCa cells to the growth inhibitory and apoptotic effects of docetaxel. Importantly, deletions of ATP7B in mPCa tissues predict significantly better survival of patients after their first chemotherapy than those with wild-type ATP7B (P = 0.0006). In addition, Disulfiram (DSF), an FDA approved drug for treatment of alcohol dependence, in combination with copper, significantly enhanced the in vivo antitumor effects of docetaxel in a docetaxel resistant xenograft tumor model. Our analyses also revealed that DSF and copper engaged with ATP7B to decrease protein levels of COMM domain-containing protein 1 (COMMD1), S-phase kinase associated protein 2 (Skp2) and clusterin and markedly increase protein expression of cyclin-dependent kinase inhibitor 1 (p21/WAF1). Taken together, our results suggest that ATP7B is a clinically relevant and key target for improving the efficacy of docetaxel for treatment of mPCa. Citation Format: Liankun Song, Vyvyan Nguyen, Jun Xie, Matthew Tippin, Le TP Truong, Christopher A. Blair, Beverly Wang, Edward Uchio, Xiaolin Zi. ATPase copper transporting beta (ATP7B) contributes to acquired docetaxel resistance in human prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1687.

Abstract 3949: CYR61, a member of the CCN protein family, regulates IGF-1 in metastatic prostate cancer

Abstract The cysteine-rich angiogenic inducer 61 (CYR61) is a member of the cellular communication network (CCN) protein family that can be induced by growth factors. CYR61-specific functions are dependent on cellular context. In prostate cancer (PCa), CYR61 contributes to cell growth and survival through its interactions with extracellular ligands, such as integrins avb3 and a6b4, in the extracellular matrix space. Interestingly, CYR61 contains an insulin-like growth factor-binding protein domain suggesting that it may interact with insulin-like growth factor-I (IGF-1). High serum levels of IGF-1 are directly linked to PCa development and recently have been studied as a predictor of metastatic disease. Given the important roles that IGF-1 and CYR61 play in PCa and their potential interaction, it is critical to investigate their molecular interplay. This study is designated to specifically determine and characterize the molecular interaction between CYR61 and IGF-1 in metastatic PCa and to determine if this interaction contributes to aggressive tumor properties (e.g., proliferation, tumorsphere formation). Using immunoblotting, we demonstrated that CYR61 is upregulated in the metastatic cell line PC3 and downregulated in the docetaxel-resistant cell line PC3-DR. Furthermore, optimized knockdown of CYR61 using siRNA significantly reduced PC3 cell proliferation, viability, and prostasphere formation. To examine the underlying mechanisms associated with IGF-1 signaling, we assessed the activation of either the PI3/Akt and MAPK pathways in PC3 cells with CYR61 silencing. CYR61 siRNA-mediated knockdown decreased activation of the PI3/Akt pathway but did not affect the MAPK pathway. We also initiated studies to determine the co-localization of CYR61 and IGF-1 using immunofluorescence microscopy (IF). Based on the initial IF observations, we will proceed to examine if their interaction is primarily intra- or extra-cellular. For intracellular interactions, we will co-immunoprecipitate CYR61 with IGF-1 and for extracellular interactions, we will use the AVidity-based EXtracellular Interaction Screen (AVEXIS) system. Our goal is to establish the contribution of CYR61-IGF1 protein-protein interaction to PCa cell proliferation. Specifically, the correlation between PCa aggressiveness and circulating levels of CYR61 and IGF-1 is poorly studied. Their combined role may explain a proportion of aggressive PCa. Citation Format: Greisha L. Ortiz-Hernandez, Mya Walker, Leanne Woods-Burnham, Rick A. Kittles, Carlos A. Casiano, Susan L. Neuhausen. CYR61, a member of the CCN protein family, regulates IGF-1 in metastatic prostate cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3949.

KIF14 mediates cabazitaxel-docetaxel cross-resistance in advanced prostate cancer by promoting AKT phosphorylation

Docetaxel is a first-line chemotherapy drug for castration-resistant prostate cancer (CRPC); yet, some CRPC patients develop docetaxel drug resistance. Cabazitaxel is approved in the post-docetaxel treatment setting. However, recent studies suggested cross-resistance between the development of drug resistance and current treatments. In this study, we used docetaxel-resistant cell lines DU145/DTX50 and PC-3/DTX30 to measure the responses to cabazitaxel. Our findings demonstrated that docetaxel resistance could lead to cross-resistance to cabazitaxel. After docetaxel-resistant cells were treated with cabazitaxel, transcriptome analysis was performed, and the results were analyzed in combination with survival analysis and correlation analysis with Gleason score to screen the cross-resistance genes. The continuously increased expression of kinesin family member 14 (KIF14) was identified as the main cause of cross-resistance to cabazitaxel in docetaxel-resistant cells. Silencing the expression of KIF14 could restore the sensitivity of resistant PCa cells to docetaxel and cabazitaxel, attenuate proliferation and promote apoptosis of the resistant PCa cells. Notably, the depressed expression of KIF14 inhibited the phosphorylation of Akt located downstream. In summary, KIF14 mediates the cross-resistance between docetaxel and cabazitaxel, and targeting KIF14 could be an effective measurement for reversing docetaxel or cabazitaxel chemotherapy failure or enhancing the anti-tumor effects of docetaxel or cabazitaxel.

306 (PB086) - Reversal of resistance to docetaxel and cabazitaxel in prostate cancer cells with ritonavir

Background: The taxanes docetaxel and cabazitaxel are indicated for treatment of metastatic castration-resistant prostate cancer (mCRPC). Resistance to docetaxel often develops during treatment, which can be due to an increased efflux of docetaxel via P-glycoprotein (P-gp) from the intracellular site of action. Ritonavir is an inhibitor of both cytochrome P4503A4 (CYP3A4) and P-gp. In this study we investigated whether ritonavir could improve the efficacy of docetaxel through inhibition of P-gp in docetaxel-resistant prostate cancer cells. Methods: Prostate cancer cell lines DU-145 and 22Rv1 were exposed to increasing doses of docetaxel to generate resistant sublines, DU145-DOC10 and 22Rv1DOC8 respectively. Gene expression of the resistant clones was compared to that of the parental cell lines. The parental cell lines and the docetaxel resistant cell lines were treated with several drug combinations in an 8*2 matrix 3D tumor clonogenic assay. The following drug combinations were tested: docetaxel/ritonavir, docetaxel/elacridar and cabazitaxel/ritonavir. The IC50 values of single agent drugs and drug combinations as well as combination effects between drugs (using a Bliss independence dose-response evaluation) were determined in all cell lines. Results: The most differentially upregulated gene in the resistant cell lines was ABCB1 (P-gp). Western blotting showed that both resistant cell lines expressed more P-gp than the parental cell lines. A comparison of the observed docetaxel and cabazitaxel IC50 values has been summarized in the below table.Table(abstract: 306 (PB086))DU-145DU-145DOC10DU-145DOC1022Rv122Rv1DOC822Rv1DOC8Docetaxel IC50 (nM)2.99.52.8*with 10 μM ritonavir,1.354.63.0**with 32 μM ritonavir.Cabazitaxel IC50 (nM)0.61.50.6*with 10 μM ritonavir,0.79.50.7**with 32 μM ritonavir.* with 10 μM ritonavir,** with 32 μM ritonavir. Open table in a new tab We found that docetaxel resistance conferred cross-resistance to cabazitaxel. With the addition of 10 μM ritonavir (DU-145DOC10) and 32 μM ritonavir (22Rv1DOC8) the cytoxicity of docetaxel and cabazitaxel returned to the level of observed in the parental cell lines. A similar experiment with the specific P-gp inhibitor elacridar showed that a concentration of 0.3 μM was sufficient to reverse docetaxel resistance to the same level as in the parental cell lines, confirming this as an important mechanism-of-action. In DU-145-DOC10 and 22Rv1DOC8 cells strong synergistic effects were seen of the docetaxel/ritonavir as well as the cabazitaxel/ritonavir combination, in contrast to the lack of synergy seen in the parental cell lines with these combinations. Conclusions: We demonstrated the potential for the combination of docetaxel and ritonavir to reverse or offset resistance to docetaxel (and, by implication due to its cross-resistance, also to cabazitaxel), primarily mediated through inhibition of P-gp. This provides a strong rationale to clinically test these taxanes in combination with ritonavir in patients with mCRPC. Conflict of interest: Ownership: Jos Beijnen is a shareholder of Modra Pharmaceuticals. Board of Directors: Colin Freund and Eric van der Putten are directors of Modra Pharmaceuticals.

Abstract 5332: Prostate cancer targeting therapy using PSA promoter-driven perforin expression vector encapsulated in liposomes conjugated with anti-PSMA antibody

Abstract Perforin secreted from cytotoxic T cells and natural killer cells play important roles in anti-tumor immunoreaction leading to cancer cell apoptosis. The aim of this study is to investigate whether perforin-rich tumor microenvironment could inhibit tumor growth. Advanced prostate cancer (PC) expresses high amount of prostate-specific antigen (PSA) that is a possible therapeutic target, therefore, we constructed a PSA promoter-driven perforin expression vector for its predominant expression in tumor microenvironment of PC. Prostate membrane-specific antigen (PSMA) expressed on PC cell surface is also an attractive therapeutic target. To deliver perforin expression vector to PC, it was encapsulated in liposomes conjugated with anti-PSMA antibody (PSMA-pLipo), then the anti-tumor effect of PSMA-pLipo was evaluated using docetaxel-resistant PC cell line, 22Rv1DR, expressing both PSA and PSMA. The conjugated anti-PSMA antibody on PSMA-pLipo recognized recombinant PSMA protein, and incubation with PSMA-pLipo induced perforin expression in 22Rv1DR cells but not in PC-3 cells not expressing both PSA and PSMA. Growth of 22Rv1DR and PC-3 cells was barely inhibited by PSMA-pLipo alone. Perforin functions in concert with cytotoxic lymphocytes, therefore anti-tumor effect of PSMA-pLipo was analyzed in the presence of human peripheral blood mononuclear cells (PBMCs). Low concentration of PSMA-pLipo with human PBMCs significantly inhibited growth of 22Rv1DR cells but not PC-3 cells. High concentration of PSMA-pLipo with human PBMCs showed nearly complete inhibition of 22Rv1DR cell growth. In a mouse xenograft model implanted with 22Rv1DR cells, PSMA-pLipo was intravenously administrated via tale vein and tumor volume was monitored. PSMA-pLipo inhibited growth of 22Rv1DR cells compared to injection of anti-PSMA antibody alone. The data presented here suggest that gene therapy expressing perforin specifically in PC cells could be a novel therapy for advanced PC. Citation Format: Kosuke Mizutani, Kyojiro Kawakami, Yasunori Fujita, Taku Kato, Manabu Takai, Daiki Kato, Koji Iinuma, Takuya Koie, Masafumi Ito. Prostate cancer targeting therapy using PSA promoter-driven perforin expression vector encapsulated in liposomes conjugated with anti-PSMA antibody [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5332.

Itraconazole Reverts ABCB1-Mediated Docetaxel Resistance in Prostate Cancer.

Docetaxel (DTX) was the first chemotherapeutic agent to demonstrate significant efficacy in the treatment of men with metastatic castration-resistant prostate cancer. However, response to DTX is generally short-lived, and relapse eventually occurs due to emergence of drug-resistance. We previously established two DTX-resistant prostate cancer cell lines, LNCaPR and C4-2BR, derived from the androgen‐dependent LNCaP cell line, and from the LNCaP lineage-derived androgen-independent C4-2B sub-line, respectively. Using an unbiased drug screen, we identify itraconazole (ITZ), an oral antifungal drug, as a compound that can efficiently re-sensitize drug-resistant LNCaPR and C4-2BR prostate cancer cells to DTX treatment. ITZ can re-sensitize multiple DTX-resistant cell models, not only in prostate cancer derived cells, such as PC-3 and DU145, but also in docetaxel-resistant breast cancer cells. This effect is dependent on expression of ATP-binding cassette (ABC) transporter protein ABCB1, also known as P-glycoprotein (P-gp). Molecular modeling of ITZ bound to ABCB1, indicates that ITZ binds tightly to the inward-facing form of ABCB1 thereby inhibiting the transport of DTX. Our results suggest that ITZ may provide a feasible approach to re-sensitization of DTX resistant cells, which would add to the life-prolonging effects of DTX in men with metastatic castration-resistant prostate cancer.

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.

EIF4A3-Induced circARHGAP29 Promotes Aerobic Glycolysis in Docetaxel-Resistant Prostate Cancer through IGF2BP2/c-Myc/LDHA Signaling

Upregulation of a novel circRNA, circARHGAP29, promotes docetaxel resistance and glycolytic metabolism, suggesting it could be a prognostic biomarker and therapeutic target in chemoresistant prostate cancer.

Combination treatment of docetaxel with caffeic acid phenethyl ester suppresses the survival and the proliferation of docetaxel-resistant prostate cancer cells via induction of apoptosis and metabolism interference

BackgroundDocetaxel has been approved by USFDA as a first-line treatment for castration-resistant prostate cancer (CRPC) patients. Patients receiving androgen deprivation therapy along with docetaxel result in superior survival, lower serum prostate specific antigen (PSA) level, and better quality of life. However, a significant proportion of these patients ultimately develop resistance to docetaxel within months. Caffeic acid phenethyl ester (CAPE), one of the main bioactive components extracted from the propolis, has been reported to be effective for repressing the tumor growth, the migration and invasion of prostate cancer (PCa) cells, as well as the downstream signaling and stability of androgen receptor (AR). We hence determined if combination treatment of docetaxel with CAPE can suppress the proliferation and the survival of docetaxel-resistant PCa cells.MethodsWe established docetaxel-resistant PC/DX25 and DU/DX50 CRPC cell lines from PC-3 and DU-145 human PCa cells, respectively. Proliferation assay, MTT assay, flow cytometry with Annexin V staining, Comet Assay, and nude mice xenograft model were applied to determine the effects of combination treatment on cell proliferation and survival of the docetaxel-resistant PCa cells. Micro-Western Array (MWA) and qRT-PCR were used to investigate the molecular mechanism lying underneath.ResultsCombination treatment effectively suppressed the proliferation, survival and tumor growth of docetaxel-resistant PCa cells both in vitro and in nude mice. Comet assay and flow cytometry indicated that combination treatment induced apoptosis in docetaxel-resistant PCa cells. MWA and Western blotting assay revealed that combination treatment suppressed protein expression of Bcl-2, AKT2, c-Myc, apoptosis and caspase activation inhibitor (AVEN), pyruvate kinase M2 (PKM2) but increased protein expression of Bax, caspase 3, cytochrome c, glucose-6-phosphate dehydrogenase (G6PD) and acylglycerol kinase (AGK). Overexpression of Bcl-2 in the docetaxel-resistant PCa cells enhanced cell proliferation of docetaxel-resistant PCa cells under combination treatment. Analysis with qRT-PCR suggested that combination treatment decreased cholesterol biosynthesis genes DHCR24 (24-dehydrocholesterol reductase) and LSS (lanosterol synthase) but increased genes involved in glycolysis and TCA cycle.ConclusionsCombination treatment of docetaxel with CAPE effectively suppressed the proliferation and survival of docetaxel-resistant PCa cells via inhibition of Bcl-2 and c-Myc as well as induction of metabolism interference. Combination treatment can be beneficial for patients with docetaxel-resistant PCa.