Prostate Cancer Cell Lines Research Articles

New cyano-acrylamide derivatives incorporating the thiophene moiety: Synthesis, anti-cancer, gene expression, DNA fragmentation, DNA damage, and in silico studies

New cyano-acrylamide derivatives bearing the thiophene structure were prepared and rigorously characterized. Thus, the reaction of diethyl 5-(2-cyanoacetamido)-3-methylthiophene-2,4-dicarboxylate with aldehydes in the presence of piperidine afforded the title compounds in very good to excellent yields (range 81-96%, mean 91%). All the newly synthesized compounds were evaluated as anti-cancer agents against five human cancer cell lines namely, MCF7 (human Caucasian breast adenocarcinoma), PC3 (human prostate cancer cell line), HepG2 (Human hepatocellular carcinoma), PACA2 (epithelial cell line derived from tumor tissue of pancreas), and BJ1 (normal skin fibroblast). While most of the compounds showed good activity against MCF-7, they showed limited activity against PC3, HepG2, and PACA2. Compound 9 showed potent anti-cancer activity against the PACA2 cell line with IC50 131.18 µM compared with the reference drug (doxorubicin, IC50 52.06 µM), while compound 10 exhibited potent anti-cancer activity against HePG2 cell lines with IC50 24.51 µM compared to the positive control (doxorubicin, IC50 39.73 µM). The gene expression, DNA fragmentation, and DNA damage of the most promising compounds were discussed. Additionally, compounds 9 and 10 showed favorable binding energies and strong interactions with CDK2, EGFR, ERα, VEGFFR, and Topoisomerase II receptors in the molecular docking simulation. These results suggest that these compounds have the potential to be effective candidates for anti-cancer therapy. Additionally, the in-silico ADMET profiles indicated that these compounds adhere to the Lipinski rules, indicating favorable physicochemical properties. These findings further support their potential for continued drug development efforts.

Brucine Suppresses Malignant Progression of Prostate Cancer by Decreasing Sarcosine Accumulation via Downregulation of GNMT in the Glycine/sarcosine Metabolic Pathway.

Prostate cancer (PCa) remains a leading cause of cancer-related incidence and mortality in men. Disruptions in amino acid (AA) metabolism contribute to the disease progression, with brucine, a glycine antagonist, exhibiting antitumor effects. This study explores the antitumor impact of brucine on PCa and investigates its mechanisms in regulating AA metabolic pathways. The study employed the PCa cell line DU-145, characterized by high sarcosine (Sar) levels, for various assays including Cell Counting Kit-8 (CCK8), wound healing, Transwell, 5-Ethynyl-2'-deoxyuridine (EDU), TdT mediated dUTP Nick End Labeling (TUNEL), flow cytometry, Western blot, and ultra-high-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Network pharmacological analysis determined the anticancer mechanisms of brucine. Sar levels in DU-145 cells were significantly higher than in normal prostatic epithelial cells RWPE-1. Treatment with brucine resulted in a marked decrease in cell viability, proliferation, invasion, and migration, while promoting apoptosis in a dose-dependent manner. Sar levels decreased with increasing brucine concentration. Network pharmacology analysis linked brucine's anticancer effect to the AA metabolism and glycine N-methyltransferase (GNMT) pathways. GNMT expression in prostate cancer tissues and The Cancer Genome Atlas database was significantly elevated compared to controls. Treatment with brucine led to downregulation of GNMT expression in DU-145 cells without significant effect on sarcosine dehydrogenase (SARDH). Addition of recombinant GNMT partially reversed the inhibitory effects of brucine on DU-145 cells. Treatment with brucine downregulates GNMT expression in DU-145 cells, reducing Sar accumulation and inhibiting tumor progression. These findings provide new insights into the antitumor mechanisms of brucine in PCa.

Neuroendocrine differentiation (ND) in sensitivity of neuroendocrine tumor (NET) cells to ONC201/TIC10 cancer therapeutic.

Prostate cancer (PCa) neuroendocrine tumor (NET)-like cells with low or absent androgen receptor (AR) signaling cause hormone therapy resistance and poor prognosis. Small cell lung carcinoma (SCLC), a high-grade NET, presents with metastasis early and has poor survival. ONC201/TIC10 is a first-in-class cancer therapeutic with clinical activity in diffuse gliomas and neuroendocrine tumors. We hypothesized that markers of neuroendocrine differentiation, activation of the integrated stress response (ISR) and the TRAIL pathway, as well as the expression of ClpP, contribute to neuroendocrine tumor cell death and sensitivity to ONC201. We show that PCa and SCLC cell lines (N=6) are sensitive to ONC201, regardless of the extent of neuroendocrine differentiation. Endogenous levels of some NET markers (CgA, FoxO1, ENO2, PGP9.5, SOX2) are present in a spectrum in PCa and SCLC cell lines. Overexpression of neural transcription factor BRN2 in DU145 PCa cells does not increase expression of NET differentiation markers FoxO1, ENO2, PGP9.5, and CgA at 48 hours. However, the transient BRN2 overexpression showed slight decreases in some NET markers on the spectrum while maintaining sensitivity of PCa cells to ONC201 before any phenotypic change related to NET differentiation. Our results show that ONC201 has preclinical activity against PCa including those without NET markers or in PCa cells with transient overexpression of neural transcription factor BRN2. Our results have relevance to activity of ONC201 in PCa where most castrate-resistant androgen-independent cancers are not therapy resistant due to NET differentiation. Importantly, NET differentiation does not promote resistance to ONC201 supporting further clinical investigations across the spectrum of PCa.

Neuroendocrine Prostate Cancer Drivers SOX2 and BRN2 Confer Differential Responses to Imipridones ONC201, ONC206, and ONC212 in Prostate Cancer Cell Lines.

Prostate cancer (PCa) is the leading cause death from cancer in men worldwide. Approximately 30% of castrate-resistant PCa's become refractory to therapy due to neuroendocrine differentiation (NED) that is present in <1% of androgen-sensitive tumors. First-in-class imipridone ONC201/TIC10 has shown clinical activity against midline gliomas, neuroendocrine tumors and PCa. We explored the question of whether NED promotes sensitivity to imipridones ONC201 and ONC206 by inducible overexpression of SOX2 and BRN2, well-known neuroendocrine drivers, in human PCa cell lines DU145 or LNCaP. Slight protection from ONC201 or ONC206 with SOX2 and BRN2 overexpression was observed in the inducible LNCaP cells but not in the DU145 cells. At 2 months, there was an apparent increase in CLpP expression in LNCaP SOX2-overexpressing cells but this did not confer enhanced sensitivity to ONC201. DU145 SOX2-overexpressing cells had a significantly reduced ONC201 sensitivity than DU145 control cells. The results support the idea that treatment of castrate-resistant prostate cancer by imipridones may not be significantly impacted by neuroendocrine differentiation as a therapy-resistance mechanism. The results support further testing of imipridones across subtypes of androgen-sensitive and castrate-resistant prostate cancer.

Design, Synthesis, and Biological Evaluation of Potent EZH2/LSD1 Dual Inhibitors for Prostate Cancer.

As histone modification enzymes, EZH2 mediates H3K27 trimethylation (H3K27me3), whereas LSD1 removes methyl groups from H3K4me1/2 and H3K9me1/2. Synergistic anticancer effects of combining inhibitors of these two enzymes are observed in leukemia and prostate cancer. Thus, a series of EZH2/LSD1 dual inhibitors are designed and synthesized to evaluate their anticancer activity. After the structure-activity study, one of the best compounds, ML234, displayed excellent antiproliferative capacity against prostate cancer cell lines LNCAP, PC3, and 22RV1. Enzymatic assays ascertained that the anticancer potency of ML234 was mediated through coinhibition of EZH2 and LSD1. Moreover, the accumulation of H3K4me2 and H3K9me2 and the decrease of H3K27me3 induced by ML234 were verified by Western blot analysis. More importantly, the compound remarkably suppressed the tumor growth and enhanced the therapeutic efficacy of clinical drug enzalutamide in the 22RV1 xenograft mouse model, indicating that it may have potential as an anticancer agent in prostate cancer.

Role of Kindlin 2 in prostate cancer

Kindlin-2 is a cytoskeletal adapter protein that is present in many different cell types. By virtue of its interaction with multiple binding partners, Kindlin-2 intercalates into numerous signaling pathways and cytoskeletal nodes. A specific interaction of Kindlin-2 that is of paramount importance in many cellular responses is its direct binding to the cytoplasmic tails of integrins, an interaction that controls many of the adhesive, migratory and signaling responses mediated by members of the integrin family of cell-surface heterodimers. Kindlin-2 is highly expressed in many cancers and is particularly prominent in prostate cancer cells. CRISPR/cas9 was used as a primary approach to knockout expression of Kindlin-2 in both androgen-independent and dependent prostate cancer cell lines, and the effects of Kindlin-2 suppression on oncogenic properties of these prostate cancer cell lines was examined. Adhesion to extracellular matrix proteins was markedly blunted, consistent with the control of integrin function by Kindlin-2. Migration across matrices was also affected. Anchorage independent growth was markedly suppressed. These observations indicate that Kindlin-2 regulates hallmark features of prostate cancer cells. In androgen expressing cells, testosterone-stimulated adhesion was Kindlin-2-dependent. Furthermore, tumor growth of a prostate cancer cell line lacking Kindlin-2 and implanted into the prostate gland of immunocompromised mice was markedly blunted and was associated with suppression of angiogenesis in the developing tumor. These results establish a key role of Kindlin-2 in prostate cancer progression and suggest that Kindlin-2 represents an interesting therapeutic target for treatment of prostate cancer.

MED12 and CDK8/19 Modulate Androgen Receptor Activity and Enzalutamide Response in Prostate Cancer.

Prostate cancer progression is driven by androgen receptor (AR) activity, which is a target for therapeutic approaches. Enzalutamide is an AR inhibitor that prolongs the survival of patients with advanced prostate cancer. However, resistance mechanisms arise and impair its efficacy. One of these mechanisms is the expression of AR-V7, a constitutively active AR splice variant. The Mediator complex is a multisubunit protein that modulates gene expression on a genome-wide scale. MED12 and cyclin-dependent kinase (CDK)8, or its paralog CDK19, are components of the kinase module that regulates the proliferation of prostate cancer cells. In this study, we investigated how MED12 and CDK8/19 influence cancer-driven processes in prostate cancer cell lines, focusing on AR activity and the enzalutamide response. We inhibited MED12 expression and CDK8/19 activity in LNCaP (AR+, enzalutamide-sensitive), 22Rv1 (AR-V7+, enzalutamide-resistant), and PC3 (AR-, enzalutamide-insensitive) cells. Both MED12 and CDK8/19 inhibition reduced cell proliferation in all cell lines, and MED12 inhibition reduced proliferation in the respective 3D spheroids. MED12 knockdown significantly inhibited c-Myc protein expression and signaling pathways. In 22Rv1 cells, it consistently inhibited the AR response, prostate-specific antigen (PSA) secretion, AR target genes, and AR-V7 expression. Combined with enzalutamide, MED12 inhibition additively decreased the AR activity in both LNCaP and 22Rv1 cells. CDK8/19 inhibition significantly decreased PSA secretion in LNCaP and 22Rv1 cells and, when combined with enzalutamide, additively reduced proliferation in 22Rv1 cells. Our study revealed that MED12 and CDK8/19 regulate AR activity and that their inhibition may modulate response to enzalutamide in prostate cancer.

Prostate Cancer's Silent Partners: Fibroblasts and Their Influence on Glutamine Metabolism Manipulation.

Cancer-associated fibroblast (CAF)s in the tumour microenvironment (TME) modulate the extracellular matrix, interact with cancer cells, and facilitate communication with infiltrating leukocytes, significantly contributing to cancer progression and therapeutic response. In prostate cancer (PCa), CAFs promote malignancy through metabolic rewiring, cancer stem cell regulation, and therapy resistance. Pre-clinical studies indicate that targeting amino acid metabolism, particularly glutamine (Gln) metabolism, reduces cancer proliferation and stemness. However, most studies lack the context of CAF-cancer interaction, focusing on monocultures. This study assesses the influence of CAFs on PCa growth by manipulating Gln metabolism using colour-labelled PCa cell lines (red) and fibroblast (green) in a co-culture system to evaluate CAFs' effects on PCa cell proliferation and clonogenic potential. CAFs increased the proliferation of hormone-sensitive LNCaP cells, whereas the castration-resistant C4-2 cells were unaffected. However, clonogenic growth increased in both cell lines. Gln deprivation and GLS1 inhibition experiments revealed that the increased growth rate of LNCAP cells was associated with increased dependence on Gln, which was confirmed by proteomic analyses. Tissue analysis of PCa patients revealed elevated GLS1 levels in both the PCa epithelium and stroma, suggesting that GLS1 is a therapeutic target. Moreover, the median overall survival analysis of GLS1 expression in the PCa epithelium and stroma identified a "high-risk" patient group that may benefit from GLS1-targeted therapies. Therefore, GLS1 targeting appears promising in castration-resistant PCa patients with high GLS1 epithelium and low GLS1 stromal expression.

Impact of Cryopreserved Placental Allografts on Biochemical Recurrence in Prostate Cancer.

Human placental allografts are widely used to promote wound healing. Placental (or amniotic membrane/umbilical cord) allografts are placed along the neurovascular bundles during radical prostatectomy to improve continence and erectile function recovery. It is unknown whether placental allografts impact biochemical recurrence (BCR). This was a single-surgeon retrospective study of 566 robotic radical prostatectomies performed from April 2015 to March 2021. The patients were divided into three groups: the negative control, Brand A, and Brand B. Brand A and Brand B were both cryopreserved amniotic membrane (CAM) allografts. A total of 324 cases were included for BCR Kaplan-Meier and risk-adjusted multivariate analyses (362 for continence analysis). In vitro analyses were performed to determine the effect of CAM allografts on prostate cancer (PCa) cell line growth. For propensity score-matched analysis (adjusting for pre-operative PSA, tumor stage, Gleason Grade, and margin status), (1) the allograft groups did not show differences in time to BCR vs. the negative control group (p = 0.7), and (2) combined allograft treatment groups showed better continence recovery vs. the negative controls (p = 0.01). In vitro, placental allografts reduced PCa cell line growth in co-culture assays. cryopreserved AM allografts (combined or individual brands) did not show a significant effect on BCR but improved continence recovery for PCa patients.

Chemical composition of essential and fixed oils of Tagetes erecta fruits (Iran) and their implications in inhibition of cancer signaling

The current research was conducted to explore, for the first time, Tagetes erecta L. (family Asteraceae) fruits from northwest Iran in terms of the chemical composition of essential and fixed oils, their cytotoxic activities, and the inhibitory effect of essential oil on the PI3K/AKT/mTOR signaling pathway. The volatile oil was obtained through hydrodistillation (Clevenger apparatus). According to gas chromatography–mass spectrometry analysis, the essential oil was rich in cyclic monoterpenoids, 2-isopropyl-5-methyl-3-cyclohexen-1-one (19.99%), d-limonene (12.75%), terpinolene (11.64%) and also the saturated fatty acid palmitic acid (19.09%). Furthermore, the seeds of T. erecta were extracted using hexane by the maceration method. The analysis of fatty acid profile of the fixed oil by gas chromatography-flame ionization detector (GC-FID) demonstrated that the most predominant fatty acids in fixed oil were linoleic acid (59.53%), palmitic acid (13.70%), stearic acid (10.20%), and oleic acid (9.20%). The cytotoxic activity of essential oil, crude oil, and fraction A (obtained from fixed oil) were evaluated by using the MTT assay on MCF7 (human breast cancer cell line), PC3 (human prostate cancer cell line), and U87MG (human glioblastoma cell line). Finally, the effect of essential oil on inhibiting the PI3K/Akt/mTOR signaling pathway was evaluated using real-time PCR. The essential oil exhibited vigorous cytotoxic activity on the U87MG cell line, with an IC50 value of 32.65 μg/mL. Interestingly, the essential oil significantly inhibited the PI3K/AKT/mTOR cascade compared to the non-treated group. Our results suggest that the essential oil holds promise as an anticancer agent for glioblastoma cell lines. To the best of our knowledge, this study is the first to report on the profile of the essential oil of T. erecta fruits and its implications for targeting the PI3K/AKT/mTOR signaling pathway.

Un-methylation of NUDT21 represses docosahexaenoic acid biosynthesis contributing to enzalutamide resistance in prostate cancer

AimsThe recent approval of enzalutamide for metastatic castration-sensitive prostate cancer underscores its growing clinical significance, raising concerns about emerging resistance and limited treatment options. While the reactivation of the androgen receptor (AR) and other genes plays a role in enzalutamide resistance, identifications of novel underlying mechanism with therapeutic potential in enzalutamide-resistant (EnzaR) cells remain largely elusive. MethodsDrug-resistant prostate cancer cell lines, animal models, and organoids were utilized to examine NUDT21 function by transcriptomic and metabolomic analyses through loss-of-function and gain-of-function assays. Notably, a mono-methylation monoclonal antibody and conditional-knockin transgenic mouse model of NUDT21 were generated for evaluating its function. ResultsNUDT21 overexpression acts as a crucial alternative polyadenylation (APA) mediator, supported by its oncogenic role in prostate cancer. PRMT7-mediated mono-methylation of NUDT21 induces a shift in 3’UTR usage, reducing oncogenicity. In contrast, its un-methylation promotes cancer growth and cuproptosis insensitivity in EnzaR cells by exporting toxic copper and suppressing docosahexaenoic acid (DHA) biosynthesis. Crucially, NUDT21 inhibition or DHA supplementation with copper ionophore holds therapeutic promise for EnzaR cells. ConclusionsThe un-methylation of NUDT21-mediated 3’UTR shortening unveils a novel mechanism for enzalutamide resistance, and our findings offer innovative strategies for advancing the treatment of prostate cancer patients experiencing enzalutamide resistance.

Targeting PLOD2 suppresses invasion and metastatic potential in radiorecurrent prostate cancer

BackgroundMetastatic relapse of prostate cancer after radiotherapy is a significant cause of prostate cancer-related morbidity and mortality. PLOD2 is a mediator of invasion and metastasis that we identified as being upregulated in our highly aggressive radiorecurrent prostate cancer cell line.MethodsPatient dataset analysis was conducted using a variety of prostate cancer cohorts. Prostate cancer cell lines were treated with siRNA, or the drug PX-478 prior to in vitro invasion, migration, or in vivo chick embryo (CAM) extravasation assay. Protein levels were detected by western blot. For RNA analysis, RNA sequencing was conducted on PLOD2 knockdown cells and validated by qRT-PCR.ResultsPLOD2 is a negative prognostic factor associated with biochemical relapse, driving invasion, migration, and extravasation in radiorecurrent prostate cancer. Mechanistically, HIF1α upregulation drives PLOD2 expression in our radiorecurrent prostate cancer cells, which is effectively inhibited by HIF1α inhibitor PX-478 to reduce invasion, migration, and extravasation. Finally, the long non-coding RNA LNCSRLR acts as a promoter of invasion downstream of PLOD2.ConclusionsTogether, our results demonstrate for the first time the role of PLOD2 in radiorecurrent prostate cancer invasiveness, and point towards its potential as a therapeutic target to reduce metastasis and improve survival outcomes in prostate cancer patients.

METTL3-mediated m6A modification of circGLIS3 promotes prostate cancer progression and represents a potential target for ARSI therapy

BackgroundCircular RNAs (circRNAs) have been shown to be involved in tumorigenesis and progression. However, the role of circGLIS3 (hsa_circ_0002874) in prostate cancer (PCa) has yet not been reported.MethodsCandidate circRNA were determined through comprehensive analysis of public datasets, PCa cell lines, and tissues data. A series of cellular functional assays, including CCK-8, colony formation, wound healing, and transwell assays were performed. Subsequently, RNA sequencing, RNA immunoprecipitation, methylated RNA immunoprecipitation, microRNA pulldown, luciferase reporter assay, and western blot were used to explore the underlying molecular mechanisms. Moreover, the xenograft tumor mouse model was established to elucidate the function of circGLIS3.ResultsCircGLIS3, derived from exon 2 of the parental GLIS3 gene, was identified as a novel oncogenic circRNA in PCa that was closely associated with the biochemical recurrence. Its expression levels were upregulated in PCa tissues and cell lines as well as enzalutamide high-resistant cells. The cellular functional assays revealed that circGLIS3 promoted PCa cell proliferation, migration, and invasion. METTL3-mediated N6-methyladenosine (m6A) modification maintained its upregulation by enhancing its stability. Mechanically, CircGLIS3 sponged miR-661 to upregulate MDM2, thus regulating the p53 signaling pathway to promote cell proliferation, migration, and invasion. Furthermore, in vitro and in vivo experiments, the knockdown of circGLIS3 improved the response of PCa cells to ARSI therapies such as enzalutamide.ConclusionsMETTL3-mediated m6A modification of circGLIS3 regulates the p53 signaling pathway via the miR-661/MDM2 axis, thereby facilitating PCa progression. Meanwhile, this study unveils a promising potential target for ARSI therapy for PCa.

1,3-Disubstituted thiourea derivatives: Promising candidates for medicinal applications with enhanced cytotoxic effects on cancer cells

The distinct chemical structure of thiourea derivatives provides them with an advantage in selectively targeting cancer cells. In our previous study, we selected the most potent compounds, 2 and 8, with 3,4-dichloro- and 3-trifluoromethylphenyl substituents, respectively, across colorectal (SW480 and SW620), prostate (PC3), and leukemia (K-562) cancer cell lines, as well as non-tumor HaCaT cells. Our research has demonstrated their anticancer potential by targeting key molecular pathways involved in cancer progression, including caspase 3/7 activation, NF-κB (Nuclear Factor Kappa-light-chain-enhancer of activated B cells) activation decrease, VEGF (Vascular Endothelial Growth Factor) secretion, ROS (Reactive Oxygen Species) production, and metabolite profile alterations. Notably, these processes exhibited no significant alterations in HaCaT cells. The effectiveness of the studied compounds was also tested on spheroids (3D culture). Both derivatives 2 and 8 increased caspase activity, decreased ROS production and NF-κB activation, and suppressed the release of VEGF in cancer cells. Metabolomic analysis revealed intriguing shifts in cancer cell metabolic profiles, particularly in lipids and pyrimidines metabolism. Assessment of cell viability in 3D spheroids showed that SW620 cells exhibited better sensitivity to compound 2 than 8. In summary, structural modifications of the thiourea terminal components, particularly dihalogenophenyl derivative 2 and para-substituted analog 8, demonstrate their potential as anticancer agents while preserving safety for normal cells.

Novel coumarin-6-sulfonamide-chalcone hybrids as glutathione transferase P1-1 inhibitors.

Multidrug resistance (MDR) mechanisms in cancer cells are greatly influenced by glutathione transferase P1-1 (hGSTP1-1). The use of synthetic or natural compounds as hGSTP1-1 inhibitors is considered an effective approach to overcome MDR. Nine compounds consisting of coumarin-6-sulfonamide linked to chalcone derivatives were synthesized and evaluated for their ability to inhibit hGSTP1-1. Among the synthetic derivatives, compounds 5g, 5f, and 5a displayed the most potent inhibitory effect, with IC50 values of 12.2 ± 0.5 μΜ, 12.7 ± 0.7 and 16.3 ± 0.6, respectively. Kinetic inhibition analysis of the most potent molecule, 5g, showed that it behaves as a mixed-type inhibitor of the target enzyme. An in vitro cytotoxicity assessment of 5a, 5f, and 5g against the human prostate cancer cell lines DU-145 and PC3, as well as the breast cancer cell line MCF-7, demonstrated that compound 5g exhibited the most pronounced cytotoxic effect on all tested cell lines. Molecular docking studies were performed to predict the structural and molecular determinants of 5g, 5f, and 5a binding to hGSTP1-1. In agreement with the experimental data, the results revealed that 5g exhibited the lowest docking score among the three studied inhibitors as a consequence of shape complementarity, governed by van der Waals, hydrogen bonds and a π-π stacking interaction. These findings suggest that coumarin-chalcone hybrids offer new perspectives for the development of safe and efficient natural product-based sensitizers that can target hGSTP1-1 for anticancer purposes.

In vitro and in vivo evaluation of Bombesin-MMAE conjugates for targeted tumour therapy

Targeted tumour therapy has proved to be an efficient alternative to overcome the limitations of conventional chemotherapy. The upregulation of the bombesin receptor 2 (BB2) in several malignancies and the advantages offered by peptide drug conjugates over antibody drug conjugates in terms of production and tumour targeting motivated us to synthesise and test bombesin conjugates armed with the tubulin binder monomethyl auristatin E. The widely used Val-Cit-PABC was initially included as cathepsin cleavable self-immolative linker for the release of the free drug. However, the poor stability of the Val-Cit-conjugates in mouse plasma encouraged us to consider the optimised alternatives Glu-Val-Cit-PABC and Glu-Gly-Cit-PABC. Conjugate BN-EVcM1, featuring Glu-Val-Cit-PABC, combined suitable stability (t(½) in mouse and human plasma: 8.4 h and 4.6 h, respectively), antiproliferative activity in vitro (IC50 = 29.6 nM on the human prostate cancer cell line PC-3) and the full release of the free payload within 24 h. Three conjugates, namely BN-EGcM1, BN-EVcM1 and BN-EVcM2, improved the accumulation of MMAE in PC-3 human prostate cancer xenograft mice models, compared to the administration of the free drug. Among them, BN-EVcM1 also stood out for the significantly extended survival of mice in in vivo acute efficacy studies and for the significant inhibition of the growth of a PC-3 tumour in mice in both acute and chronic efficacy studies.

Analysis of heterochromatin remodeling as a mechanism of radiotherapy (RT) resistance through molecular profiling of radioresistant (RR) head and neck (HNC) and prostate cancers (PCa).

137 Background: Resistance to RT remains a clinical challenge, given our limited understanding of the molecular pathways underpinning radioresistance. Here, we investigated for mechanisms linked to RR using paired experimental and clinico-molecular HNC and PCa datasets for discovery and validation of novel pathways that may be enriched in RR cancers. Methods: For experimental data, we generated RR HNC (FaDu, HK1) and PCa (22Rv1, DU145) cell lines following high-dose X-irradiation (90 Gy in 45 fr). Genotypic characterization of RR and wildtype (WT) cells was performed by genomic and transcriptomic sequencing using WES (100X) and RNAseq (Illumina, CA), respectively, followed by functional characterization by western blot (WB) and immunofluorescence (IF). For clinico-molecular data, we utilized two prospectively recruited RT cohorts, consisting of 311 patients with HNC (n=158) and PCa (n=153). Tumor transcriptomes were profiled using RNAseq and Affymetrix ST array (ThermoFisher, CA), respectively. Comparative analyses of molecular profiles were first performed between RR and WT in vitro models to identify dysregulated pathways that were linked to radioresistance, and subsequently tested for association with disease-free survival (DFS) in the clinical cohorts. Results: Comparative genomic analyses between RR and WT HNC and PCa models revealed an abundance of acquired mutations in the RR compared with WT models, with a higher mutation count observed in PCa than HNC cells (SNV counts: 2,158 [DU145-RR] and 1,387 [22Rv1-RR] vs 396 [FaDu-RR] and 25 [HK1-RR], P&lt;0.0001). Mutational signature analyses indicated a common enrichment of DNA mismatch repair-related mutational signatures (SBS3, SBS14, and SBS21) across the RR HNC and PCa models. Transcriptomic profiling revealed an upregulation of the BAHD1 gene, which is involved in heterochromatin formation, across the 4 RR cell lines, corroborated by an enrichment of heterochromatin-related genesets. These results were consistent with functional characterization of RR versus WT cells by WB and IF indicating increased DNA repair capacity and heterochromatin response post-4 Gy irradiation. We further confirmed the dependency of the RR phenotype on the heterochromatin response with BAHD1 knockdown, resulting in reversal of these cellular responses. Finally, our results were supported by survival analyses indicating an inferior DFS post-RT in patients with HNC and PCa harboring a higher expression of heterochromatin-related geneset (HNC: HRhigh vs low 1.51 ; PCa: HRhigh vs low 1.37 ). Conclusions: Herein, by leveraging on paired experimental and clinico-molecular datasets, we have uncovered a novel BAHD1-dependent heterochromatin response that underpins resistance to RT in HNC and PCa, which may be amendable to systemic agents targeting chromatin remodeling.

Altered expression and localization of nuclear envelope proteins in a prostate cancer cell system

BackgroundThe nuclear envelope (NE), which is composed of the outer and inner nuclear membranes, the nuclear pore complex and the nuclear lamina, regulates a plethora of cellular processes, including those that restrict cancer development (genomic stability, cell cycle regulation, and cell migration). Thus, impaired NE is functionally related to tumorigenesis, and monitoring of NE alterations is used to diagnose cancer. However, the chronology of NE changes occurring during cancer evolution and the connection between them remained to be precisely defined, due to the lack of appropriate cell models.MethodsThe expression and subcellular localization of NE proteins (lamins A/C and B1 and the inner nuclear membrane proteins emerin and β-dystroglycan [β-DG]) during prostate cancer progression were analyzed, using confocal microscopy and western blot assays, and a prostate cancer cell system comprising RWPE-1 epithelial prostate cells and several prostate cancer cell lines with different invasiveness.ResultsDeformed nuclei and the mislocalization and low expression of lamin A/C, lamin B1, and emerin became more prominent as the invasiveness of the prostate cancer lines increased. Suppression of lamin A/C expression was an early event during prostate cancer evolution, while a more extensive deregulation of NE proteins, including β-DG, occurred in metastatic prostate cells.ConclusionsThe RWPE-1 cell line-based system was found to be suitable for the correlation of NE impairment with prostate cancer invasiveness and determination of the chronology of NE alterations during prostate carcinogenesis. Further study of this cell system would help to identify biomarkers for prostate cancer prognosis and diagnosis.

CDK9 inhibition constrains multiple oncogenic transcriptional and epigenetic pathways in prostate cancer

BackgroundCyclin-dependent kinase 9 (CDK9) stimulates oncogenic transcriptional pathways in cancer and CDK9 inhibitors have emerged as promising therapeutic candidates.MethodsThe activity of an orally bioavailable CDK9 inhibitor, CDKI-73, was evaluated in prostate cancer cell lines, a xenograft mouse model, and patient-derived tumor explants and organoids. Expression of CDK9 was evaluated in clinical specimens by mining public datasets and immunohistochemistry. Effects of CDKI-73 on prostate cancer cells were determined by cell-based assays, molecular profiling and transcriptomic/epigenomic approaches.ResultsCDKI-73 inhibited proliferation and enhanced cell death in diverse in vitro and in vivo models of androgen receptor (AR)-driven and AR-independent models. Mechanistically, CDKI-73-mediated inhibition of RNA polymerase II serine 2 phosphorylation resulted in reduced expression of BCL-2 anti-apoptotic factors and transcriptional defects. Transcriptomic and epigenomic approaches revealed that CDKI-73 suppressed signaling pathways regulated by AR, MYC, and BRD4, key drivers of dysregulated transcription in prostate cancer, and reprogrammed cancer-associated super-enhancers. These latter findings prompted the evaluation of CDKI-73 with the BRD4 inhibitor AZD5153, a combination that was synergistic in patient-derived organoids and in vivo.ConclusionOur work demonstrates that CDK9 inhibition disrupts multiple oncogenic pathways and positions CDKI-73 as a promising therapeutic agent for prostate cancer, particularly aggressive, therapy-resistant subtypes.

Targeting adenocarcinoma and enzalutamide‑resistant prostate cancer using the novel anti‑androgen inhibitor ADA‑308.

Prostate cancer (PCa) is the leading cause of cancer‑related death among men worldwide. PCa often develops resistance to standard androgen deprivation therapy and androgen receptor (AR) pathway inhibitors, such as enzalutamide (ENZ). Therefore, there is an urgent need to develop novel therapeutic strategies for this disease. The efficacy of ADA‑308 was evaluated through in vitro assessments of AR activity and cell proliferation, alongside in vivo studies. ADA‑308 has emerged as a promising candidate, demonstrating potent inhibition of AR‑sensitive adenocarcinoma as well as ENZ‑resistant PCa cell lines. The results of the study revealed that ADA‑308 effectively blocked AR activity, including its nuclear localization, and inhibited cell proliferation in vitro. Furthermore, ADA‑308 demonstrated notable efficacy in vivo, with a robust antitumor response in ENZ‑resistant models. These findings establish the role of ADA‑308 as a potent AR inhibitor that overcomes resistance to AR‑targeted therapies and highlights its potential as a novel therapeutic approach in advanced PCa management.