Activation of neural lineage networks and ARHGEF2 in enzalutamide-resistant and neuroendocrine prostate cancer and association with patient outcomes.

Background: Treatment-emergent neuroendocrine prostate cancer (NEPC) after androgen receptor (AR) targeted therapies is an aggressive variant of prostate cancer with an unfavorable prognosis. The underlying mechanisms for early neuroendocrine differentiation are poorly defined and diagnostic and prognostic biomarkers are needed.
Methods: We performed transcriptomic analysis on the enzalutamide-resistant prostate cancer cell line C4-2B MDVR and NEPC patient databases to identify neural lineage signature (NLS) genes. Correlation of NLS genes with clinicopathologic features was determined. Cell viability was determined in C4-2B MDVR and H660 cells after knocking down ARHGEF2 using siRNA. Organoid viability of patient-derived xenografts was measured after knocking down ARHGEF2.
Results: We identify a 95-gene NLS representing the molecular landscape of neural precursor cell proliferation, embryonic stem cell pluripotency, and neural stem cell differentiation, which may indicate an early or intermediate stage of neuroendocrine differentiation. These NLS genes positively correlate with conventional neuroendocrine markers such as chromogranin and synaptophysin, and negatively correlate with AR and AR target genes in advanced prostate cancer. Differentially expressed NLS genes stratify small-cell NEPC from prostate adenocarcinoma, which are closely associated with clinicopathologic features such as Gleason Score and metastasis status. Higher ARGHEF2, LHX2, and EPHB2 levels among the 95 NLS genes correlate with a shortened survival time in NEPC patients. Furthermore, downregulation of ARHGEF2 gene expression suppresses cell viability and markers of neuroendocrine differentiation in enzalutamide-resistant and neuroendocrine cells. Conclusions: The 95 neural lineage gene signatures capture an early molecular shift toward neuroendocrine differentiation, which could stratify advanced prostate cancer patients to optimize clinical treatment and serve as a source of potential therapeutic targets in advanced prostate cancer.

Introduction

Next-generation antiandrogen agents such as enzalutamide and abiraterone have been developed to suppress androgen biogenesis and block the activated AR signaling[1-4]. However, in recent years, it has become more prevalent for NEPC occurrence in CRPC patients who have received potent AR signaling inhibitors, which trigger the AR-independent tumor growth and neuroendocrine differentiation[5-7]. In neuroendocrine differentiation cancers such as small-cell lung cancer, various studies have demonstrated that transcriptional plasticity contributes to intratumoral heterogeneity and treatment resistance[8-10]. Prostate cancer cells upon the AR-targeted treatment acquire stem-like characteristics through tumor cell plasticity and molecular reprogramming, which contribute to transdifferentiation of neuroendocrine[11].

Neuroendocrine prostate cancer (NEPC) is a lethal phenotype of prostate cancer, characterized by immunobiological expression of NE markers including synaptophysin (SYP), chromogranin (CHGA), low expression of prostate-specific antigen (PSA), and androgen receptor (AR), and unresponsiveness to standard hormonal targeted treatments. Neuroendocrine differentiation gives rise to a more aggressive and malignant clinical phenotype, which has a higher incidence under the exposure to AR-directed therapies in prostate cancer patients[5,12-14]. Patients diagnosed with NEPC usually have dismal survival time and unfavorable prognosis[15,16]. The median survival for t-NEPC is approximately 20 months[14]. Given the rapid progression and malignancy of NEPC, there is an urgent need for early detection of neuroendocrine differentiation in CRPC patients for clinical diagnosis and therapeutic decision making.

Dissecting treatment-induced tumor heterogeneity and identifying biomarkers of neuroendocrine differentiation led to the successful translation to clinical diagnosis, prognosis, and therapies for prostate cancer patients. Recent studies have identified gene signatures regulating cell cycle progression as a prognostic predictor in NEPC[5,17]. In aggressive NEPC, a set of neuronal-related genes (BSN, CRMP1, GPRIN1, INA, MAST1, MYT1, RAB3C, SNAP25, UNC13A) were identified that confer to resistance to contemporary antiandrogen therapies[18]. Comparing AR-high metastatic CRPC, AR low, amphicrine and neuroendocrine PCa, 26-gene transcriptional signatures (including CELF3, PCSK1, SOX2, POU3F2, LMO3, NKX2-1, etc.) has been developed to distinguish variant phenotypes of metastatic castration-resistant prostate cancer (mCRPC)[19]. In different types of cancers, biomarker-targeted therapies have dramatically improved clinical outcomes. Therefore, the identification and validation of early emergent neural lineage markers that govern the response to antiandrogen resistances and neuroendocrine differentiation represents a fundamental unmet need.

In this study, we assessed the neural lineage pathway in enzalutamide-resistant C4-2B MDVR cell line and NEPC patients. We identified 95 gene neural lineage signatures (NLSs) underlying the molecular landscape of neural precursor cell proliferation, embryonic stem cell pluripotency, and neural stem cell differentiation/projection, which may represent a spectrum of neuroendocrine differentiation in advanced PCa. These NLS genes positively correlate with classical NE makers such as NSE, SYP, and CHGA and negatively correlate with AR and AR target genes in small-cell neuroendocrine group compared with prostate adenocarcinoma. Differentially expressed NLS genes can distinguish small-cell/NEPC from adenocarcinoma, indicating a diagnostic potential for CRPC patient stratification. Kaplan–Meier survival analyses revealed that the levels of ARHGEF2, LHX2, and EPHB2 expression among the 95 neural lineage genes were associated with shortened survival time. Further studies showed that the downregulation of ARHGEF2 gene expression suppresses cell viability and markers of neuroendocrine in enzalutamide-resistant and neuroendocrine cells. Together, these findings suggest that treatment-emergent NEPC has a unique neural lineage network that can potentially serve as prognostic markers and can be targeted to treat enzalutamide-resistant and NEPC.

Methods

Cell culture

C4-2B parental and enzalutamide-resistant (referred to as C4-2B MDVR) prostate cancer cells were obtained from the American Type Culture Collection (ATCC) and were cultured in RPMI 1640 supplemented with 10% fetal bovine serum (FBS), 100 units/ml penicillin and 0.1 mg/ml streptomycin. C4-2B MDVR cells were maintained in 20 μM enzalutamide for over 10 months. Enzalutamide purchased from Selleck Chemicals was dissolved in DMSO and stored at −20 °C. C4-2B parental cells were passaged alongside the resistant cells as the corresponding control. H660 cells were obtained from ATCC and were cultured in RPMI 1640 plus 5% FBS as the basic medium and were supplemented with 0.005 mg/ml insulin, 0.01 mg/ml transferrin, 30 nM sodium selenite, 10 nM hydrocortisone, 10 nM beta-estradiol, extra 2 mM L-glutamine. All cells were cultured in 37 °C humidified incubators with 5% CO2.

Cell transfection

C4-2B MDVR and H660 cells were plated at 60–80% confluence in 60 mm dishes and transfected with 25 nM of small interfering RNA (siRNAs) targeting ARHGEF2 sequence (Catalog# 31215495), LHX2 sequence (Catalog# 312154951), EPHB2 sequence (Catalog# 312154948), or control siRNA (Catalog# 12935300) using Lipofectamine RNAiMAX transfection reagent (Invitrogen). The effects of siRNA knocking down were evaluated using qRT-PCR and western blot after 48 h transfection.

RNA extraction and RNA sequencing

Total RNA was isolated from C4-2B parental, MDVR cells by TriZOL reagent (Invitrogen). RNA integrity was assessed using RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system (Agilent Technologies). RNA sequencing libraries were generated using NEBNext Ultra RNA Library Prep Kit for Illumina (NEB, USA) according to the manufacturer’s instruction and index codes were added to attribute sequences to each sample. cDNA fragments of preferentially 150–200 bp in length were selected using AMPure XP system (Beckman Coulter, USA). The index-coded samples were further clustered using PE Cluster Kit cBot-HS (Illumina) and sequenced on the Illumina platform. Paired-end clean reads were aligned to the reference human genome assembly (GRCh38/hg38) using the Spliced Transcripts Alignment to a Reference software. The gene expression level was calculated as FPKM (fragments per kilobase of transcript sequence per Millions base pairs sequenced). The hierarchical clustering analysis was used to cluster samples with similar expression patterns for different phenotypes of prostate cancer tumors.

Real-time quantitative RT-PCR

Total RNA was extracted from C4-2B parental, MDVR, H660 cells, LuCaP35CR, and LuCaP49 patient-derived xenograft (PDX) tumors using TriZOL reagent (Invitrogen Catalog# #15596018). cDNA was prepared using reverse transcriptase purchased from Promega. The cDNAs were subjected to quantitative PCR using SsoFast EvaGreen Supermix (Bio-Rad) according to the manufacturer’s manual. Samples were run in triplicates on Bio-Rad CFX-96 real-time cycler. Each reaction was normalized by coamplification of GAPDH. Primers used for real-time PCR are as follows: ARHGEF2, 5′-TACCTGCGGCGAATTAAGAT-3′ (forward) and 5′-AAACAGCCCGACCTTCTCTC-3′ (reverse); LHX2, 5′-CCGCACACTTCAACCATGC-3′ (forward) and 5′-CCACGCCATTGTAGTAGGGAA-3′ (reverse); EPHB2, 5′-CGAGCCACGTTACATCA-3′ (forward) and 5′-TCAGTAACGCCGTTCACAGC-3′ (reverse); GAPDH, 5′-ATGAGTCCTTCCACGATACCA-3′ (forward) and 5′-GAAATCCCATCACCATCTTCC-3′ (reverse).

Cell lysate collection and western blotting

Cells were harvested and lysed in RIPA buffer and the concentration was determined by Coomassie Plus Protein Assay (Thermo Scientific, Catlog#1856210). Protein extracts were resolved by SDS-PAGE, and proteins were transferred to nitrocellulose membranes. After blocking in 5% milk in PBS-0.1% Tween-20 for 1 h at room temperature, targeted proteins were detected by incubating the NC membrane with primary antibodies at 4 °C overnight. AR (441), 1:1000 dilution (Santa Cruz Biotechnology, Santa Cruz, CA); GEF-H1 (55B6) Rabbit mAb from Cell Signaling Technology (Catalog#4076); CHGA from Santa Cruz Biotechnology (Catalog#393941); SYP from Santa Cruz Biotechnology (Catalog#9116); GAPDH from Cell Signaling Technology (Catalog#2118). GAPDH was used as loading control. Following secondary antibody incubation, immunoreactive proteins were visualized by applying Immobilon Crescendo Western HRP substrate (Millipore, Catalog#WBLUR0500).

Gene Set Enrichment Analysis

Gene expression results were subjected to Gene Set Enrichment Analysis (GSEA) to determine patterns of pathway activity in different group of samples. GSEA using GSEA software from the Broad Institute was used to identify biological function pathways based on Molecular Signature Database. The pathway analysis was performed using pathway gene sets annotated by Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes, and PathCards (Pathway Unification Database) (https://pathcards.genecards.org/). Pathways enriched with normalized enrichment score (NES) and a nominal p value lower than 0.05 and FDR q value lower than 0.25 were considered to be significant.

PDX tumor xenografts and organoid culture

All animals used in this study received humane care in compliance with applicable ethnic regulations, policies, and guidelines relating to animals. Animal experiments were conducted in accordance with animal protocols approved by the Institutional Animal Care and Use Committee of the University of California Davis. Male C.B17/lcrHsd-Prkdc-SCID mice from ENVIGO were used for the PDX xenograft at 4–6 weeks of age and housed in standard cages in ultraclean barrier facilities. The housing conditions for the mice were as follows: 12-h light-dark cycle; temperature was 68–79 °F; and humidity was 30–70%. Animals demonstrating sickness or severe stress were euthanized and excluded. Otherwise, all data were included in the study.

LuCaP35 CR and LuCaP49 PDX models were obtained from the University of Washington and established at the UC Davis. Four- to six-week-old male C.B17/lcrHsd-Prkdc-SCID mice (ENVIGO) were surgically castrated and implanted subcutaneously into the flanks with approximately 2 mm3 LuCaP35 CR tumor pieces after 1 week. LuCaP49 tumor pieces were directly implanted subcutaneously to 4–6-week-old male C.B17/lcrHsd-Prkdc-SCID mice (ENVIGO) and tumors were collected after 5 weeks of growth. LuCaP35CR and LuCaP49 PDX tumor specimens were collected from the corresponding donor mice and subjected to RT-qPCR and organoid culture.

LuCaP PDX-derived tumor tissues were collected and washed twice in cold PBS, and subjected to dissection to 2–4 mm3 with scalpel blade. Tumors were digested using collagenase IV (STEMCELL) in a 60-mm2 petri dish and incubated in 37 °C humidified incubators with 5% CO2 for 15–30 min until tumor cells were dispersed in the digestive medium. Advanced DMEM medium supplemented with 1X GlutaMAX (Gibco), 1 M HEPES (Gibco) and 100 u/ml penicillin and 0.1 mg/ml streptomycin were added to the cell suspension, and then filtered through 40-μm cell strainers to obtain single cell suspension. The cells were centrifuged at 500 g for 3 min and the pellet was resuspended in an advanced DMEM complete medium containing GlutaMAX (Gibco), 100 units/ml penicillin, 0.1 mg/ml streptomycin, B27 (Gibco), N-Acetylcysteine (Thermo Scientific), Human Recombinant EGF (Thermo Scientific), Recombinant FGF-10 (Invitrogen), A-83-01 (Tocris), SB202190 (Bioscience), Nicotinamide (Thermo Scientific), dihydrotestosterone (Sigma), PGE2 (Bioscience), Noggin (Thermo Scientific) and R-spondin (R&D Systems)[20,21]. Tumor cell pellet was seeded in 96-well plate with Matrigel diluted with 1:3 ratio of ADMEM complete medium and incubated in 37 °C humidified incubators with 5% CO2 for 15 min to solidify the 3D Matrigel complex. Then ADMEM complete medium mixed with corresponding treatment was added to each well. Viability of the organoids was analyzed using CellTiter Glo Luminescent assay (Promega) and visualized by immunofluorescence using LIVE/DEAD® Viability/Cytotoxicity Assay Kit (Thermo Scientific) according to the manufacturer’s protocol.

Statistics and reproducibility

A two-sided Wilcoxon test was used to analyze the significance of gene expression between two groups. A p value less than 0.05 was considered significant (*p < 0.05, **p < 0.01, ***p < 0.001) unless otherwise indicated. Kaplan–Meier curves were plotted to analyze the survival probability of the two groups. A log-rank test was used to compare the overall survival or disease-free survival between the two groups of different gene expression level. A p value lower than 0.05 indicated that the two groups differed significantly in overall survival or disease-free survival.