Menin inhibition suppresses castration-resistant prostate cancer and enhances chemosensitivity

Abstract

Disease progression and therapeutic resistance of prostate cancer (PC) are linked to multiple molecular events that promote survival and plasticity. We previously showed that heat shock protein 27 (HSP27) acted as a driver of castration-resistant phenotype (CRPC) and developed an oligonucleotides antisense (ASO) against HSP27 with evidence of anti-cancer activity in men with CRPC. Here, we show that the tumor suppressor Menin (MEN1) is highly regulated by HSP27. Menin is overexpressed in high-grade PC and CRPC. High MEN1 mRNA expression is associated with decreased biochemical relapse-free and overall survival. Silencing Menin with ASO technology inhibits CRPC cell proliferation, tumor growth, and restores chemotherapeutic sensitivity. ChIP-seq analysis revealed differential DNA binding sites of Menin in various prostatic cells, suggesting a switch from tumor suppressor to oncogenic functions in CRPC. These data support the evaluation of ASO against Menin for CRPC.

Fig. 1. Menin as a novel HSP27 ubiquitin-proteasome regulated partner in CRPC.

A HSP27, Menin, and vinculin protein levels analyzed were by western blot in CSPC (LNCaP-Mock) and CRPC (LNCaP-HSP27) models. B Menin and GAPDH western blot in CSPC (LNCaP) and AIPC (DU-145, PC-3) cell lines. C LNCaP-Mock, LNCaP-HSP27, and PC-3 cells were fixed and immunostained using Menin (red immunofluorescence) and HSP27 (green immunofluorescence) antibodies. Yellow puncta represented colocalization. D LNCaP-Mock and LNCaP-HSP27 cell lysates were used to immunoprecipitate (IP) Menin using mouse anti-Menin or mouse anti-immunoglobulin (IgG) antibodies. Total cell lysate (TCL) represents proteins from LNCaP-Mock versus LNCaP-HSP27 cells were extracted and blotted with anti-Menin or anti-HSP27 antibodies. E PC-3 cells were treated with OGX-427 or OGX-control for two days and then harvested for protein extraction and tested by western blot analysis using an anti-Menin, anti-HSP27, and anti-vinculin antibodies. F Menin and GAPDH levels were analyzed by quantitative reverse transcription-PCR (qRT-PCR) on total RNAs extracted from a culture of PC-3 treated with OGX-427 or OGX-control. Menin mRNA levels were analyzed after normalization to GAPDH RNA levels. Results are expressed as the percentage of PC-3 cells transfected with the OGX-control (100%). G Protein lysates from LNCaP-Mock and LNCaP-HSP27 cells were used to immunoprecipitate (IP) Menin, followed by western blotting with an anti-ubiquitin (Ub) antibody. H, I PC-3 cells were treated with OGX-427 or OGX-control for 2 days. After transfection, cells were harvested for protein extraction or pretreated with cycloheximide (10 μg/ml) followed by MG-132 (10 μmol/l) for 48 h and tested by western blot analysis using an anti-Menin, anti-HSP27, and anti-vinculin antibodies. HSP27, heat shock protein 27.

Fig. 2. Menin expression correlates with prostate cancer progression, hormone resistance, and associated with poor prognosis.

A Representative microscopic fields of Menin immunostaining in Gleason tissue microarray (TMA). B Means of Menin expression staining in Gleason TMA composed from 198 patients (2 cores from each patient): 41 BHP patients (SEM: 0.056) and 157 PC patients in different Gleason G1 (SEM: 0.073), G2 (SEM: 0.071), G3 (SEM: 0.068), G4 (SEM: 0.075), and G5 (SEM 0.080). C Representative microscopic fields of Menin immunostaining in Neoadjuvant Hormonal Therapy (NHT) TMA. D Means of Menin expression in NHT TMA composed from 94 patients (2 cores from each patient): 38 no treated patients (naïve) (SEM: 0.034), 21 patients treated with NHT (SEM: 0.043), and 35 patients CRPC (SEM: 0.078). Specimens were graded from 0 to +3 intensity, representing the range from no staining to heavy staining by visual scoring and automated quantitative image analysis by Image-pro Plus software. E Box-plot of MEN1 mRNA expression levels in 272 “normal” samples (normal tissue and prostate benign hypertrophia), 1643 PC primary tumor samples, and 90 PC metastatic samples collected from public data sets. The p-values are for the Student t-test. F Similar to (E), but for 3 hormone-sensitive versus 63 hormone-resistant metastatic samples. G Kaplan–Meier biochemical relapse-free survival (BRFS) and H overall survival (OS) curves in the “MEN1-low” class (green curve) versus “MEN1-high” class (red curve). The p-value is for the log-rank test.

Fig. 3. Menin-ASO causes 90% abrogation of Menin expression.

A PC-3 cells were treated with 100 nM of differents ASOs or control-ASO. Two days after the second transfection, proteins were extracted and analyzed by western blot. B Bands were quantified using ImageJ software normalized to GAPDH protein levels. Data were normalized to control-ASO (100%). C PC-3 cells were treated with indicated concentrations of ASOs or control-ASO. Two days after the second transfection, proteins were extracted and analyzed by western blot. D Bands were quantified using ImageJ software normalized to GAPDH protein levels. Data were normalized to untreated-control (100%).

Fig. 4. Menin is involved in cytoprotection induced by HSP27 and Menin-ASO inhibits PC cell growth and restores treatment sensitivity.

A LNCaP-Mock and LNCaP-HSP27 cells were treated with 100 nM Menin-ASO or control-ASO. After 2 days, docetaxel was added for 24 h. Cell viability was determined using the MTT test. Data are shown as mean ± SEM, n = 3. Two-tailed, unpaired Student’s t-test (****P ≤ 0.0001; ***P ≤ 0.001; **P ≤ 0.01). B PC-3 cells were transfected with 100 nM Menin-ASO or control-ASO for two days and treated with docetaxel for 24 h. Cell viability was determined using the MTT test. Data were normalized to untreated cells, and are shown as mean ± SEM, n = 3. Two-tailed, unpaired Student’s t-test (***P ≤ 0.001; **P ≤ 0.01). C PC-3 cells were transfected with 100 nM Menin-ASO or control-ASO for 2 days after the second transfection. Apoptosis was assessed by Annexin V binding. Flow cytometry was used to quantify the apoptotic rates (D) and to quantify the percentage of PC-3 cells in each cell cycle phase. Data were normalized to untreated cells and are shown as mean ± SEM, n = 3. Two-tailed, unpaired Student’s t-test (***P ≤ 0.001). E Western blot analysis of Menin expression in PC-3 xenografts after 7 days of treatment with Menin- or control-ASO. F Mice bearing AIPC (PC-3) tumors were randomly selected for treatment with Menin- or control-ASO. When PC-3 tumors reached 50 mm³, 12.5 mg/kg/mouse of Menin- or control-ASO were injected intraperitoneally (i.p.) daily for 5 weeks in animals receiving ASO monotherapy. Tumor volume was measured once weekly and calculated by the formula length × width × depth × 0.5236. Data show mean ± SEM. Two-tailed, unpaired Student’s t-test, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. G Ki-67 IHC staining of tumor tissues to access tumor cell proliferation.

Fig. 5. Functional profiles comparison between normal prostate PNT1A (N), LNCaP (ASPC), and PC-3 (AIPC) models.

A Venn diagram showing the differential of Menin gene binding in PNT1A (N), LNCaP (ASPC), and PC-3 (AIPC) cells. Enrichment of differential bound genes (DBGs) of Menin in PNT1A (N) (172) versus PC LNCaP (2509) and PC-3 (1384) models B by KEGG pathways C by GO terms enrichment of DBGs related to the molecular functions (MF). D GO terms enrichment of DBGs using the exclusive list Menin targets genes in LNCaP (ASPC) (2509) versus PC-3 (AIPC) (1384) models related to the biological process (BP). E KEGG pathways enrichment of DBGs using the full list Menin targets genes in LNCaP (ASPC) (11127) versus PC-3 (AIPC) (10113). GeneRatio = amount of DBGs enriched in the pathway/amount of all genes in the annotation gene set, p.adjust ≤ 0.01. GO, gene ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes. F Box-plot of the PC1 metagene score based upon expression of Menin target genes of each resistance pathway in 3 hormone-sensitive versus 63 hormone-resistant metastatic samples. The p-values are for the Student t-test. G Kaplan–Meier overall survival curves in 99 patients with mCRPC treated with a first-line next-generation ARSI (abiraterone or enzalutamide) from the Sawyers group’s dataset [29] according to high (red curve) versus low (black curve) PC1 metagene score based upon Menin target genes of the Platinum resistance pathway (natural cut-off = 0). The p-value is for the log-rank test.

Fig. 6. ChIP-seq and RNA-seq comparative analyses and biological validation.

A Scatter plots showing differential MEN1 ChIP-seq targets between ASPC (LNCaP) and AIPC (PC-3) models. Peaks with a fold change (FC) > 2 were considered as upregulated peaks (red). Peaks with FC < 0.5 were considered as downregulated peaks (blue). B Cross plot between ChIP-seq and RNA-seq comparative analyses. Yellow dots represent genes significantly modulated p ≤ 0.05, FC > 2. C KEGG and Reactome pathways analysis of Menin-peaks nearby genes. Grayscale values illustrate gene enrichment (i.e., number of genes observed/expected) for each KEGG or Reactome pathways. The red scale indicates the P-value (–log10) and greyscale represents gene percent (i.e., % of genes observed/total number of genes within each KEGG or Reactome pathways). D Validation of pathways activated by Menin in the AIPC model. PC-3 cells were treated with 100 nM of Menin- or control-ASO and 2 days after second transfection proteins were extracted and analyzed by western blot. E PC-3 cells were transfected with 100 nM Menin-ASO or control-ASO for 2 days and treated at 27.9 μM of Cisplatin for 48 h after the second transfection. Cell viability was determined using the MTT test. Data were normalized to untreated cells, and are shown as mean ± SEM, n = 3. Two-tailed, unpaired Student’s t-test (***P ≤ 0.001).

Fig. 7. Menin co-regulators.

A Identification of Menin interacting transcription factors performed by bioinformatics analysis of IP-MS Menin partners list (Normal Prostate Cell for PNT1A, Androgen Independent Prostate Cancer Cell). B PNT1A, LNCaP, and PC3 cell lysates were used to immunoprecipitate (IP) Menin using anti-Menin or anti-immunoglobulin (IgG) antibodies. Total cell lysate (TCL) were extracted and blotted with anti-Menin, anti-SNW1, or anti-ERH antibodies.

Fig. 8. Menin switches from tumor suppressor to oncogenic role in prostate cancer.

Schematic display illustrating how Menin switches from tumor suppressor role through miRNA regulation in normal prostate to oncogenic role in the AIPC AR-negative model, through activation of PI3K/AKT pathways and drive disease progression to CRPC and resistance to treatments (created using BioRender).