Plexin D1 emerges as a novel target in the development of neural lineage plasticity in treatment-resistant prostate cancer

Treatment-induced neuroendocrine prostate cancer (t-NEPC) often arises from adenocarcinoma via lineage plasticity in response to androgen receptor signaling inhibitors, such as enzalutamide. However, the specific regulators and targets involved in the transition to NEPC are not well understood. Plexin D1 (PLXND1) is a cellular receptor of the semaphorin (SEMA) family that plays important roles in modulating the cytoskeleton and cell adhesion. Here, we found that PLXND1 is highly expressed and positively correlated with neuroendocrine markers in patients with NEPC. High PLXND1 expression is associated with poorer prognosis in prostate cancer patients. Additionally, PLXND1 was upregulated and negatively regulated by androgen receptor signaling in enzalutamide-resistant cells. Knockdown or knockout of PLXND1 inhibit neural lineage pathways, suppressing NEPC cell proliferation, PDX tumor organoid viability, and xenograft tumor growth. Mechanistically, the chaperone protein HSP70 regulates PLXND1 protein stability through degradation, and inhibition of HSP70 decreases PLXND1 expression and NEPC organoid growth. In summary, our findings suggest that PLXND1 could be a new therapeutic target and molecular indicator for NEPC.


Introduction
Prostate cancer is the most common cancer in males and the fth leading cause of cancer-related deaths worldwide [1,2].The primary treatments for early stage and localized prostate cancer include radical prostatectomy and radical radiotherapy.In the United States, survival rates for localized, regional, and metastatic stages have been reported to be 100%, 96.1%, and 18.5%, respectively [3].Androgen deprivation therapy (ADT) is a standard treatment for advanced prostate cancer.However, despite initial remission, the disease often progresses to castration-resistant prostate cancer (CRPC) [4].
Androgen receptor (AR) signaling inhibitors such as enzalutamide, abiraterone, and apalutamide have been developed for the treatment of CRPC [5,6].Unfortunately, patients undergoing this treatment often develop resistance via various mechanisms [7].This resistance can lead to the emergence of neuroendocrine differentiation and its features [8].Following AR-targeted therapy, prostate cancer cells undergo changes in their characteristics, resembling stem cell-like properties, due to tumor cell plasticity and molecular reprogramming.This transformation contributes to neuroendocrine trans-differentiation [9].Neuroendocrine prostate cancer (NEPC) is a speci c subset of CRPC that is resistant to AR signaling inhibitors.NEPC is characterized by low or absent AR expression, independence from AR signaling, and acquisition of a neuroendocrine phenotype [10].De novo NEPC is rare, accounting for less than 2% of all primary prostate cancers [11].Treatment-induced NEPC has been reported in 10-17% of patients with advanced therapy-resistant CRPC [12].The survival rate of NEPC patients is poor, with a 5-year survival rate of only 10% [10,13].To maximize the survival bene ts of patients with NEPC, a comprehensive understanding of the molecular mechanisms involved in NEPC development and progression is crucial.Plexin D1 (PLXND1) is a member of the plexin family of transmembrane proteins that plays an essential role in axonal guidance and vascular patterning [14].Normally, PLXND1 expression is low in adult tissues and is associated with a subset of activated broblasts and macrophages [15].Studies have reported the upregulation of PLXND1 in various tumors, including pancreatic, melanoma, ovarian, colon, and prostate tumors [14,16].However, there are currently no reports available on the expression and function of PLXND1 in NEPC.
In this study, we found that enzalutamide not only activated neural lineage pathways but also induced the upregulation of PLXND1.The analysis indicated that PLXND1 was upregulated in NEPC samples and was associated with poor patient prognosis.Further investigation revealed that AR negatively regulates PLXND1 in enzalutamide-resistant prostate cancer cells.In vitro and in vivo studies have suggested that inhibition of PLXND1 could suppress NEPC.Additionally, the study identi ed that the HSP70 inhibition has the potential to degrade the protein expression of PLXND1.Overall, this study proposed that PLXND1 could serve as a prognostic marker and a potential target for treating NEPC.

Western blot analysis
Whole-cell protein extracts were separated by SDS-PAGE and transferred onto nitrocellulose membranes.

Co-immunoprecipitation assay
Equal amounts of cell lysates (1500 µg) were subjected to immunoprecipitation overnight using 1 µg of speci c antibodies, such as HSP70, along with 100 µL of protein A/G agarose with continuous rotation.
The immunoprecipitants were washed twice with 1 mL 10 mM HEPES (pH 7.9), 1mM EDTA, 150 mM NaCl, and 1% Nonidet P-40.The precipitated proteins were eluted with 60 µL SDS-PAGE sample buffer by boiling for 10 min.The eluted proteins were separated on a 6% SDS-PAGE gel, transferred to nitrocellulose membranes, and incubated with the speci ed antibodies.
Cell growth assay C4-2B-MDVR and CWR22Rv1 cells were plated in 12-well plates at a density of 1 × 10^4 cells/well in RPMI 1640 medium supplemented with 10% FBS and subjected to various treatments.H660 cells were seeded in 12-well plates at a density of 5 × 10^4 cells/well in the appropriate medium for H660 culture and were treated under various conditions.The overall cell count was used to calculate the percentage of surviving cells.

Clonogenic assay
C4-2B-MDVR and CWR22Rv1 cells were seeded at a density of 1000 cells/well in 6-well plates and exposed to various treatments for 11 d.Subsequently, the colonies were washed with PBS and stained with 0.5% crystal violet/4% formaldehyde for 30 min, and the colony count was determined.

RNA-seq data analysis
Total RNA from both control and PLXND1-knockdown C4-2B-MDVR and H660 cells was extracted using the RNeasy Mini Kit (Qiagen) and subjected to DNase digestion, according to the manufacturer's instructions.Subsequently, RNA-seq libraries were generated using 1 µg of total RNA and the Illumina TruSeq RNA Sample kit.Paired-end mRNA-Seq libraries were constructed on an Illumina HiSeq 4000 platform with 2 × 150 cycles/bases (150bp, PE), resulting in approximately 30 million reads per sample.Data analysis involved a Top Hat-Cu inks pipeline, with sequence read mapping and alignment performed using HISAT.The obtained StringTie data were then mapped and quanti ed for 27,044 unique genes/transcripts, with gene and transcript expression quanti ed as FPKM (Fragments Per Kilobase of transcript per million mapped reads).

Gene set enrichment analysis (GSEA)
GSEA was performed using Java desktop software (http://software.broadinstitute.org/gsea/index.jsp)as described previously [19].Signi cance was attributed to pathways showing enrichment with a normalized enrichment score (NES), a nominal p-value below 0.05, and an FDR q-value less than 0.25.

PDX tumor xenografts and organoid culture
Experimental procedures involving animals were approved by the Institutional Animal Care and Use Committee of UC Davis and adhered to the ARRIVE guidelines, ethical regulations, and human endpoints (animal protocol number #22246).Male 6-week-old SCID mice were procured from Envigo and housed in an animal research facility at UC Davis.To assess the impact of PLXND1 on tumor development and growth, 4 × 10^6 CWR22Rv1 cells (sgControl, sgPLXND1#1, or sgPLXND1#2) were combined in a 1:1 ratio with Matrigel (Corning) for bilateral subcutaneous injection into NSG mice.Tumor size was measured every 3-4 days with calipers, and tumor volume was calculated as width^2 × length × 0.52, starting one week after tumor inoculation.

Immunohistochemistry
Tumors were xed in formalin and para n-embedded tissue blocks were dewaxed, rehydrated, and blocked for endogenous peroxidase activity.Antigen retrieval was performed in sodium citrate buffer (0.01 mol per Litter, pH 6.0) in a microwave oven at 1000 W for 3 min and then at 200 W for 20 min.Nonspeci c antibody binding was blocked by incubation with 10% fetal bovine serum in PBS for 30 min at room temperature.Slides were then incubated with anti-PLXND1 ( 1:100, CST; or 1:200, Zen-bio) at 4°C overnight.The slides were then washed and incubated with biotin-conjugated secondary antibodies for 30 min, followed by incubation with avidin DH-biotinylated horseradish peroxidase complex for 30 min (Vectastain ABC Elite Kit, Vector Laboratories).The sections were developed using a diaminobenzidine substrate kit (Vector Laboratories) and counterstained with hematoxylin.Nuclear staining of cells was performed and the cells were counted in ve different vision elds.Images were taken using an Olympus BX51 microscope equipped with a DP72 camera.

Prostate adenocarcinoma and NEPC patient tissue samples
Primary prostate adenocarcinoma tissue microarray (n = 46) and NEPC samples (n = 3) were obtained from the Department of Urology at the West China Hospital, Sichuan University.Pathologists at the Department of Pathology, West China Hospital, Sichuan University con rmed the pathological types of prostate adenocarcinoma or NEPC.This study was approved by the Institutional Ethics Review Board of West China Hospital (No. 2017 − 324), and written informed consent was obtained from all patients.PLXND1 staining without awareness of patient clinical information by combining the area percent of positive cells from 1 to 4 (1 = 0%-25%, 2 = 26%-50%, 3 = 51%-75%, and 4 = 76%-100%) and the relative intensity of the staining gray level from 1 to 4 (1 = negative, 2 = low, 3 = medium, and 4 = high).All statistical signi cance was obtained using a z-score calculator for two population proportions (https://www.socscistatistics.com/tests/ztest/) between a group with scores of 1 and 2(negative to low intensity) and a group with scores of 3 and 4 (medium to high intensity).

Statistical analysis
Statistical analyses were performed using GraphPad Prism 9.0 (RRID:SCR_002798).Raw data were summarized by means, standard deviations (SD), and graphical summaries and then transformed, if necessary, to achieve normality.The sample size was determined based on the power to detect signi cant differences (p < 0.05).No samples or data points were excluded from the analysis.The experiments and data processing were not blinded.Data are presented as the mean ± SD from three independent experiments.Differences between individual groups were analyzed using a two-tailed Student's t-test for single comparisons or one-way analysis of variance (ANOVA), followed by the Scheffé procedure for multiple group comparisons.In the tumor growth experiments, the size of the tumor at sacri ce served as the primary response measure.Tumor growth across groups was analyzed using analysis of variance.P < 0.05 was considered statistically signi cant (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns = non-signi cant).

Data availability statement
The data obtained in this study are available upon reasonable request from the corresponding authors.

Results
PLXND1 is upregulated in NEPC and prostate cancer cells showing neural lineage plasticity.
AR signaling negatively regulates the expression of PLXND1 in enzalutamide-resistant prostate cancer.

Knockdown of PLXND1 represses the cell proliferation and improves enzalutamide treatment.
To evaluate whether PLXND1 plays a role in regulating the aggressive characteristics of neuroendocrine prostate cancer (NEPC) cells, we employed two distinct siRNAs to knockdown PLXND1 in C4-2B-MDVR, CWR22Rv1, and H660 cells, all of which exhibit neuroendocrine traits [30][31][32].The knockdown effect was validated using RT-qPCR (Fig. S3A).Subsequently, we observed that PLXND1 knockdown suppressed cell proliferation and colony formation in C4-2B-MDVR cells compared with the control groups.Similar inhibitory effects were observed in CWR22Rv1 and H660 cells (Fig. 3A-3B).Additionally, PLXND1 knockdown led to reduced expression of CDK2, CyclinA, CyclinD1, and CyclinE, along with increased expression of cleaved-PARP in C4-2B-MDVR, CWR22Rv1, and H660 cells (Fig. 3C).Importantly, we found that PLXND1 knockdown signi cantly improved enzalutamide treatment of C4-2B MDVR and CWR22Rv1 cells (Fig. 3D and Fig. S3B).Moreover, we assessed the effect of PLXND1 knockdown in an H660 organoid model.The results demonstrated that Silencing PLXND1 expression via siRNA signi cantly inhibited the viability and growth of H660 organoids (Fig. 3E).In summary, these ndings suggest that PLXND1 could potentially serve as a therapeutic target for NEPC, given its involvement in regulating key cellular processes and aggressive behavior of NEPC cells.Knockdown of PLXND1 decreases the neuroendocrine traits.
To evaluate the impact of PLXND1 knockdown on neuroendocrine traits and associated pathways, we conducted RNA-seq analysis of C4-2B-MDVR and H660 cells with silenced PLXND1.The results revealed that 287 genes were signi cantly downregulated and 349 genes were signi cantly upregulated in C4-2B-MDVR cells (Fig. 4A).Similarly, in H660 cells, 431 genes were signi cantly downregulated and 1008 genes were signi cantly upregulated (Fig. 4B).Subsequent analysis showed that six neural lineage pathways were downregulated in C4-2B-MDVR cells with PLXND1 knockdown compared to the control, including the negative regulation of axonogenesis, developmental growth, synapse assembly, neuron differentiation, neuron projection development, and neurogenesis (Fig. 4C).GO analysis also revealed the downregulation of neural lineage pathways in H660 cells transfected with siPLXND1 (Fig. S4A).Further GOBP analysis indicated that ve pathways were upregulated in C4-2B-MDVR cells with PLXND1 knockdown compared to the control, including regulation of the apoptotic signaling pathway, signal transduction by P53 class mediator, regulation of the extrinsic apoptotic signaling pathway, positive regulation of the apoptotic signaling pathway, and negative regulation of the cell cycle G1/S phase transition.KEGG analysis identi ed upregulation of the P53 signaling pathway and apoptosis in C4-2B-MDVR cells with PLXND1 knockdown compared with the control.Reactome analysis further revealed that four pathways regulating the cell cycle and apoptosis were signi cantly upregulated in C4-2B-MDVR cells with PLXND1 knockdown (Fig. 4D).GSEA analysis demonstrated that four neural lineage pathways were downregulated in C4-2B-MDVR cells with PLXND1 knockdown, including neurotransmitter uptake, regulation of receptor localization to synapses, synaptic transmission GABAergic, and regulation of GABAergic synaptic transmission (Fig. 4E).Apoptosis pathways were also upregulated in H660 cells transfected with siPLXND1 (Fig. S4B).Additionally, PLXND1 knockdown downregulated the protein expression of neuroendocrine markers, including CHGA, NSE, and SYP (Fig. 4F).Finally, heatmap analysis illustrated the downregulation of neural lineage pathway genes and the upregulation of apoptosis pathway genes in C4-2B-MDVR cells transfected with siPLXND1 (Fig. 4G).In summary, our data suggest that PLXND1 knockdown decreases neuroendocrine traits of NEPC cells.
For a protein to become functional, it requires correct folding and assembly, a process that relies on the chaperone HSP70.To determine whether HSP70 binds to PLXND1 and regulates PLXND1 expression, we conducted co-IP assays in HEK293 and CWR22Rv1 cells.The results indicated that HSP70 binds to PLXND1 in both HEK293 and CWR22Rv1 cells (Fig. 6A-6B).Subsequently, we knocked down HSP70 in CWR22Rv1 cells using siRNA, and RT-PCR and western blotting were employed to validate the knockdown e cacy (Fig. 6C-6D).The ndings revealed that HSP70 knockdown downregulated the protein expression of PLXND1 without affecting its mRNA expression of PLXND1 (Fig. 6C-6D).Using the HSP70 allosteric inhibitor JG231, we found that JG231 treatment signi cantly reduced the expression of PLXND1, CDK2, CyclinA, CyclinD1, CyclinE, and neuroendocrine markers (CHGA, NSE, and SYP) and increased the expression of cleaved-PARP and cleaved-Caspase7 in C4-2B-MDVR, CWR22Rv1, and H660 cells (Fig. 6E, Fig. S6A).To determine whether the decrease in PLXND1 protein expression induced by JG231 treatment was mediated through the proteasome pathway, we added the proteasome inhibitor MG132 to CWR22Rv1 cells.Although JG231 reduced PLXND1 protein expression, the addition of MG132 blunted the effects of JG231 (Fig. 6F).Furthermore, we investigated whether JG231 affects PLXND1 protein stability in CWR22Rv1 cells using the cycloheximide (CHX) chase assay and found that JG231 treatment signi cantly shortened the half-life of PLXND1 (Fig. 2G).Notably, JG231 treatment inhibited the growth of LuCaP49 and LuCaP93 NEPC organoids in a dose-dependent manner (Fig. 6H, Fig. S6B).Collectively, these data suggest that the chaperone protein HSP70 may control the turnover of PLXND1, and HSP70 inhibition may indirectly target PLXND1 in NEPC.

Discussion
Trans-differentiation of prostate adenocarcinoma to acquire a neuroendocrine phenotype has been extensively investigated as a crucial mechanism for the development of NEPC, a highly aggressive and therapy-resistant phenotype of prostate cancer [22,25,33].Emerging studies suggest that genes commonly upregulated in patients with a neuroendocrine phenotype regulate important neuronal functions and are associated with poor prognosis during cancer progression [22,31,32].In this study, we identi ed PLXND1 as a target of NEPC, uncovering a PLXND1-dependent mechanism that induces a lineage switch of prostate adenocarcinoma cells towards a neuroendocrine phenotype.Prior research has indicated that the invasiveness and metastasis of AR-negative prostate cancer cells (PC3 and DU145) are regulated through Notch signaling, which transcriptionally regulates PLXND1 [16].Our results indicate that PLXND1 is more highly expressed in NEPC tumors than in prostate adenocarcinoma.
We demonstrated that enzalutamide treatment induced the emergence of a neuroendocrine phenotype, including small-cell histology and positive neuroendocrine markers, consistent with published evidence [12,25,34,35].Additionally, enzalutamide therapy was found to drive the upregulation of PLXND1 in C4-2B cells treated with enzalutamide for two months.To date, there have been no published studies investigating the correlation between the upregulation of PLXND1 and the emergence of a neuroendocrine phenotype in prostate cancer treated with enzalutamide.Further analysis indicated that the expression of PLXND1 is positively correlated with classic NEPC markers such as CHGA, NSE, SYP, and CD56, and is negatively correlated with AR and its targeting genes such as KLK2, KLK3, and NKX3-1.Recent integrated bioinformatic analyses of NEPC have revealed that the expression of neurolineage genes is positively associated with NEPC markers and negatively associated with AR and its target genes [34].During the transition from prostate adenocarcinoma to NEPC and the acquisition of the neuroendocrine phenotype, the disease gradually changes from AR-dependent to AR-independent and is accompanied by the upregulation of PLXND1 expression.Furthermore, we found that AR signaling negatively regulates the expression of PLXND1 in advanced prostate cancer.PLXND1, a member of the transmembrane protein family of plexins, is essential for axonal guidance and vascular patterning [14,36].Studies on endothelial cells revealed that PLXND1 acts as a direct force sensor and collaborates with neuropilin-1 and VEGFR2 to form a mechano-complex that elicits robust and global mechanical signaling upstream of the junctional complex and integrins [37].Another study indicated that SEMA3E-PLXND1 signaling is a critical determinant of synaptic connections in sensorymotor circuits, speci c for functional and anatomical rewiring of monosynaptic connections [38].Recent evidence indicates that disruption of the axon guidance pathway mediated by SEMA3D and PLXND1 may slow the progression of pancreatic ductal adenocarcinomas [39].PLXND1 may play a key role in the process of attaining a neuroendocrine phenotype and in the progression of NEPC.Our study found that knockdown or knockout of PLXND1 inhibited the proliferation of NEPC cells and organoids and decreased the tumorigenicity of NEPC cells in vivo.PLXND1 is highly heterogeneous in different types of tumors, acting as a tumor promoter in colon cancer and ovarian endometrioid cancer, and as a tumor suppressor in breast cancer [40,41].
Currently, there are limited clinical trials on therapies for patients [42].Treatment of NEPC mainly relies on clinical trial e cacy data for small-cell lung cancer [42].Platinum-based chemotherapies, used for small cell neuroendocrine tumors, have shown response rates in the range of 10%-50% [43,44].Taxane-based and platinum-based chemotherapies are used to treat non-small cell variants of NEPC [32,44].In a previous clinical trial, 41 patients with metastatic androgen-independent prostate cancer and elevated serum CHGA or NSE levels received cisplatin and docetaxel treatment for three weeks.The results showed that 33% of these patients exhibited partial or complete responses, marked by reduced serum neuroendocrine levels, with an overall survival ranging from 1 to 38 months, with a median survival of 12 months.Nevertheless, over 50% of the treated patients experience toxic side effects, such as septic shock, asthenia, and neuropathy, highlighting the limitations of current chemotherapy-based therapies for NEPC [45].Our results suggest that the chaperone protein HSP70 regulates the expression of PLXND1 through protein degradation.HSP70 is a ubiquitous molecular chaperone that acts on a large variety of cellular protein folding and remodeling processes.It also contributes to key steps in protein degradation through the ubiquitin-proteasome system and various autophagy pathways [46,47].Previous data from our research showed that the HSP70/STUB1 complex controls the sensitivity to AR-targeted therapy in advanced prostate cancer by synergistically degrading AR and AR-V7 [48].In this study, we found that HSP70 binds to PLXND1 and regulates protein folding and degradation.Using an HSP70 inhibitor may indirectly target the PLXND1 protein and disrupt its signaling axis to improve the therapy of patients with NEPC.
Further investigation is needed to determine whether HSP70 cooperates with other proteins to degrade PLXND1, and its speci c mechanism.Additionally, previous studies have indicated that PLXND1 always binds to its ligand, SEMAs [49].SEMA-PLXND1 signaling plays important roles in cardiovascular, nervous, and immune system development as well as in cancer biology [39,41,50].One potential research direction is to explore whether PLXND1 cooperates with the SEMA family members to regulate disease progression in NEPC.
In summary, our research uncovered an important process in which PLXND1 plays a key role in gaining neuroendocrine phenotypes in prostate cancer cells, which is a critical factor in the progression of NEPC.
We suggest that targeting PLXND1 may be a promising strategy for treating NEPC.This suggests that efforts directed towards understanding and manipulating the PLXND1-driven mechanism could potentially lead to effective strategies for managing NEPC, addressing a critical need for prostate cancer supported in part by grants from the National Institutes of Health R37CA249108 (Liu), R01CA251253 (Liu), R21CA277171 (Liu), Department of Defense HT9425-23-1-0144 (Liu), HT9425-23-1-0325 (Liu), and a UC Davis Comprehensive Cancer Center Support Grant (CCSG) awarded by the National Cancer Institute (NCI P30CA093373).The maintenance and characterization of the LuCaP PDX models were supported by the Paci c Northwest Prostate Cancer SPORE (P50CA97186), Department of Defense Prostate Cancer Biorepository Network (W81XWH-14-2-0183), and National Institutes of Health P01-CA163227.Financial support: This work was supported in part by grants from NIH/NCI R37CA249108 (Liu), R01CA251253 (Liu), R21CA277171 (Liu), Department of Defense HT9425-23-1-0144 (Liu), HT9425-23-1-0325 (Liu), and UC Davis Comprehensive Cancer Center Support Grant (CCSG) awarded by the National Cancer Institute (NCI P30CA093373).

Figures
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Figure 2 AR
Figure 2

Figure 6 HSP70
Figure 6 Liu C, Lou W, Yang JC, Liu L, Armstrong CM, Lombard AP et al.Proteostasis by STUB1/HSP70 complex controls sensitivity to androgen receptor targeted therapy in advanced prostate cancer.Nat